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Abstract Hormone therapy, especially androgen deprivation therapy (ADT), is effective against prostate cancer (PC), whereas long-term ADT is a risk for metabolic/cardiovascular disorders including diabetes (DM), hypertension (HT) and dyslipidemia (DL), and might result in progression to castration-resistant prostate cancer (CRPC). We thus conducted a multicenter retrospective cohort study to ask whether CRPC progression would be associated positively with HT, DM or DL and negatively with statins prescribed for treatment of DL. In this study, 1,112 nonmetastatic PC patients undergoing ADT were enrolled. Univariate statistical analyses clearly showed significant association of HT or DM developing after ADT onset, though not preexisting HT or DM, with early CRPC progression. On the other hand, preexisting DL or statin use, but not newly developed DL or started statin prescriptions following ADT, was negatively associated with CRPC progression. Multivariate analysis revealed significant independent association of the newly developed DM or HT, or preexisting statin use with CRPC progression [adjusted hazard ratios (95% confidence intervals): 3.85 (1.65–8.98), p = 0.002; 2.75 (1.36–5.59), p = 0.005; 0.25 (0.09–0.72), p = 0.010, respectively]. Together, ADT-related development of HT or DM and preexisting statin use are considered to have positive and negative impact on CRPC progression, respectively.

Abstract Given possible involvement of the central and peripheral angiotensin system in pain processing, we conducted clinical and preclinical studies to test whether pharmacological inhibition of the angiotensin system would prevent diabetic peripheral neuropathy (DPN) accompanying type 2 diabetes mellitus (T2DM). In the preclinical study, the nociceptive sensitivity was determined in leptin-deficient ob/ob mice, a T2DM model. A clinical retrospective cohort study was conducted, using the medical records of T2DM patients receiving antihypertensives at three hospitals for nearly a decade. In the ob/ob mice, daily treatment with perindopril, an angiotensin-converting enzyme inhibitor (ACEI), or telmisartan, an angiotensin receptor blocker (ARB), but not amlodipine, an L-type calcium channel blocker (CaB), significantly inhibited DPN development without affecting the hyperglycemia. In the clinical study, the enrolled 7464 patients were divided into three groups receiving ACEIs, ARBs and the others (non-ACEI, non-ARB antihypertensives). Bonferroni’s test indicated significantly later DPN development in the ARB and ACEI groups than the others group. The multivariate Cox proportional analysis detected significant negative association of the prescription of ACEIs or ARBs and β-blockers, but not CaBs or diuretics, with DPN development. Thus, our study suggests that pharmacological inhibition of the angiotensin system is beneficial to prevent DPN accompanying T2DM.

Cav3.2, a T-type calcium channel (T-channel) family member, is expressed in the nociceptors and spinal cord, and its activity is largely suppressed by zinc under physiological conditions. In rats, intrathecal and intraplantar administration of a zinc chelator, TPEN, caused T-channel-dependent mechanical hyperalgesia, and the intraplantar, but not intrathecal, TPEN induced Cav3.2 upregulation in the dorsal root ganglion. In mice, intraplantar TPEN also caused mechanical allodynia, which was abolished by T-channel inhibitors or Cav3.2 gene deletion. Together, spinal and peripheral zinc deficiency appears to enhance Cav3.2 activity in the spinal postsynaptic neurons and nociceptors, respectively, thereby promoting pain.

Oxaliplatin often induces peripheral neuropathy, a dose-limiting adverse reaction, and in rare cases leads to sinusoidal obstruction syndrome. We thus conducted a retrospective cohort study to examine the relationship between oxaliplatin-induced peripheral neuropathy (OIPN) and hepatic impairment, and then perform a fundamental study to analyze the underlying mechanisms. Analysis of medical records in cancer patients treated with oxaliplatin indicated that laboratory test parameters of hepatic impairment including AST, ALT and APRI (AST to platelet ratio index) moderately increased during oxaliplatin treatment, which was positively correlated with the severity of OIPN (grades 1–4), and associated with later incidence of survivors with OIPN grades ≥2. In mice, hepatic injury induced by CCl4 or ethanol accelerated OIPN in mice, an effect prevented by inactivation of high mobility group box 1 (HMGB1), known to participate in OIPN, by the neutralizing antibody or thrombomodulin alfa capable of promoting its thrombin-dependent degradation. Oxaliplatin also aggravated the hepatic injury in mice. CCl4 released HMGB1 from cultured hepatic parenchymal cells, and oxaliplatin at clinically achievable concentrations released HMGB1 from hepatic parenchymal and non-parenchymal cells. Our clinical and preclinical data suggest that the development of mild hepatic impairment during oxaliplatin treatment is associated with later aggravation of OIPN.

We examined the role of ATP and high mobility group box 1 (HMGB1) in paclitaxel-induced peripheral neuropathy (PIPN). PIPN in mice was prevented by HMGB1 neutralization, macrophage depletion, and P2X7 or P2X4 blockade. Paclitaxel and ATP synergistically released HMGB1 from macrophage-like RAW264.7 cells, but not neuron-like NG108-15 cells. The paclitaxel-induced HMGB1 release from RAW264.7 cells was accelerated by co-culture with NG108-15 cells in a manner dependent on P2X7 or P2X4. Paclitaxel released ATP from NG108-15 cells, but not RAW264.7 cells. Thus, PIPN is considered to involve acceleration of HMGB1 release from macrophages through P2X7 and P2X4 activation by neuron-derived ATP.

Poly-trans-[(2-carboxyethyl)germasesquioxane] (Ge-132), an organogermanium, is hydrolyzed to 3-(trihydroxygermyl)propanoic acid (THGP) in aqueous solutions, and reduces inflammation, pain and cancer, whereas the underlying mechanisms remain unknown. Sulfides including H2S, a gasotransmitter, generated from l-cysteine by some enzymes including cystathionine-γ-lyase (CSE), are pro-nociceptive, since they enhance Cav3.2 T-type Ca2+ channel activity expressed in the primary afferents, most probably by canceling the channel inhibition by Zn2+ linked via coordinate bonding to His191 of Cav3.2. Given that germanium is reactive to sulfur, we tested whether THGP would directly trap sulfide, and inhibit sulfide-induced enhancement of Cav3.2 activity and sulfide-dependent pain in mice. Using mass spectrometry and 1H NMR techniques, we demonstrated that THGP directly reacted with sulfides including Na2S and NaSH, and formed a sulfur-containing reaction product, which decreased in the presence of ZnCl2. In Cav3.2-transfected HEK293 cells, THGP inhibited the sulfide-induced enhancement of T-type Ca2+ channel-dependent membrane currents. In mice, THGP, administered systemically or locally, inhibited the mechanical allodynia caused by intraplantar Na2S. In the mice with cyclophosphamide-induced cystitis and cerulein-induced pancreatitis, which exhibited upregulation of CSE in the bladder and pancreas, respectively, systemic administration of THGP as well as a selective T-type Ca2+ channel inhibitor suppressed the cystitis-related and pancreatitis-related visceral pain. These data suggest that THGP traps sulfide and inhibits sulfide-induced enhancement of Cav3.2 activity, leading to suppression of Cav3.2-dependent pain caused by sulfide applied exogenously and generated endogenously.

Abstract To identify risk factors for the prognosis of prostate cancer (PC), we retrospectively analyzed the impact of lifestyle-related disorders as well as PC characteristics at initial diagnosis on the progression to castration-resistant PC (CRPC) in PC patients undergoing hormone therapy. Of 648 PC patients, 230 who underwent hormone therapy and met inclusion criteria were enrolled in this study. CRPC developed in 48 patients (20.9%). Univariate analysis using Cox proportional hazard model indicated that newly developed diabetes mellitus (DM) following hormone therapy (postDM), but not preexisting DM, as well as PC characteristics at initial diagnosis including prostate-specific antigen (PSA) ≥ 18 were significantly associated with the progression to CRPC. A similar tendency was also observed in the relationship between newly developed hypertension following hormone therapy and CRPC progression. On the other hand, neither dyslipidemia nor hyperuricemia, regardless the onset timing, exhibited any association with CRPC progression. In multivariate analysis, postDM and PSA ≥ 18 were extracted as independent risk factors for CRPC progression (adjusted hazard ratios, 3.38 and 2.34; p values, 0.016 and 0.019, respectively). Kaplan–Meier analysis and log-rank test clearly indicated earlier progression to CRPC in PC patients who developed postDM or had relatively advanced initial PC characteristics including PSA ≥ 18. Together, the development of lifestyle-related disorders, particularly DM, following hormone therapy, as well as advanced PC characteristics at initial diagnosis is considered to predict earlier progression to CRPC and poor prognosis in PC patients undergoing hormone therapy.

We performed clinical retrospective study in female cancer patients and fundamental experiments in mice, in order to clarify risk factors for paclitaxel-induced peripheral neuropathy (PIPN). In the clinical study, 131 of 189 female outpatients with cancer undergoing paclitaxel-based chemotherapy met inclusion criteria. Breast cancer survivors (n = 40) showed significantly higher overall PIPN (grades 1–4) incidence than non-breast cancer survivors (n = 91). Multivariate sub-analyses of breast cancer survivors showed that 57 years of age or older and endocrine therapy before paclitaxel treatment were significantly associated with severe PIPN (grades 2–4). The age limit was also significantly correlated with overall development of severe PIPN. In the preclinical study, female mice subjected to ovariectomy received repeated administration of paclitaxel, and mechanical nociceptive threshold was assessed by von Frey test. Ovariectomy aggravated PIPN in the mice, an effect prevented by repeated treatment with 17β-estradiol. Repeated administration of thrombomodulin alfa (TMα), known to prevent chemotherapy-induced peripheral neuropathy in rats and mice, also prevented the development of PIPN in the ovariectomized mice. Collectively, breast cancer survivors, particularly with postmenopausal estrogen decline and/or undergoing endocrine therapy, are considered a PIPN-prone subpopulation, and may require non-hormonal pharmacological intervention for PIPN in which TMα may serve as a major candidate.

HMGB1, a nuclear protein, once released to the extracellular space, promotes somatic and visceral pain signals. We thus analyzed the role of HMGB1 in an intravesical substance P-induced bladder pain syndrome (BPS) mouse model. Intravesical administration of substance P caused referred hyperalgesia/allodynia in the lower abdomen and hindpaw without producing severe urothelial damage, which was prevented by an anti-HMGB1-neutralizing antibody, thrombomodulin α capable of inactivating HMGB1 and antagonists of RAGE or CXCR4. The HMGB1 inactivation or RAGE blockade also reversed the established bladder pain symptoms. HMGB1 and RAGE are thus considered to serve as therapeutic targets for BPS.

TRPV1 is phosphorylated and functionally upregulated by protein kinases, and negatively regulated by phosphatases including calcineurin. Since the clinical use of calcineurin-inhibiting immunosuppressants is commonly associated with chronic diarrhea, we examined if tacrolimus, a calcineurin inhibitor, promotes TRPV1-dependent colonic hypersensitivity in mice. Intracolonic administration of capsaicin, a TRPV1 agonist, caused referred hyperalgesia in the lower abdomen, an effect prevented by capsazepine, a TRPV1 blocker. Tacrolimus accelerated the intracolonic capsaicin-induced referred hyperalgesia. Similarly, intracolonic capsaicin caused spinal ERK phosphorylation, a marker for nociceptor excitation, an effect promoted by tacrolimus. Thus, tacrolimus may aggravate TRPV1-related colonic pain accompanying irritable bowel syndrome. Keywords: Tacrolimus, Colonic pain, TRPV1

Abstract Background Macrophage-derived high mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) protein, plays a key role in the development of chemotherapy-induced peripheral neuropathy (CIPN) caused by paclitaxel in rodents. Endothelial thrombomodulin (TM) promotes thrombin-induced degradation of HMGB1, and TMα, a recombinant human soluble TM, abolishes peripheral HMGB1-induced allodynia in mice. We thus examined whether HMGB1, particularly derived from macrophages, contributes to oxaliplatin-induced neuropathy in mice and analyzed the anti-neuropathic activity of the TM/thrombin system. Methods CIPN models were created by the administration of oxaliplatin in mice and rats, and the nociceptive threshold was assessed by von Frey test or paw pressure test. Macrophage-like RAW264.7 cells were stimulated with oxaliplatin in vitro. Proteins were detected and/or quantified by Western blotting, immunostaining, or enzyme-linked immunosorbent assay. Results Intraperitoneal administration of an anti-HMGB1-neutralizing antibody (AB) at 1 mg/kg prevented the oxaliplatin-induced allodynia in mice and rats. Antagonists of Toll-like receptor (TLR) 4, receptor for advanced glycation end products (RAGE) and CXCR4 among the HMGB1-targeted pro-nociceptive receptors, also mimicked the anti-neuropathic activity of AB in mice. Macrophage accumulation in the sciatic nerve was observed in mice treated with paclitaxel, but not oxaliplatin, and neither macrophage depletion nor inhibitors of macrophage activation affected oxaliplatin-induced allodynia. Oxaliplatin was 10- to 100-fold less potent than paclitaxel in releasing HMGB1 from macrophage-like RAW264.7 cells. Like AB, TMα at 10 mg/kg prevented the oxaliplatin-induced allodynia in mice as well as rats, an effect abolished by argatroban at 10 mg/kg, a thrombin inhibitor. The anti-neuropathic activity of TMα in oxaliplatin-treated mice was suppressed by oral anticoagulants such as warfarin at 1 mg/kg, dabigatran at 75 mg/kg, and rivaroxaban at 10 mg/kg, but not antiplatelet agents such as aspirin at 50 mg/kg and clopidogrel at 10 mg/kg. Repeated administration of the anticoagulants gradually developed neuropathic allodynia and elevated plasma HMGB1 levels in mice treated with a subeffective dose of oxaliplatin. Conclusions Our data thus suggests a causative role of HMGB1 derived from non-macrophage cells in oxaliplatin-induced peripheral neuropathy and a thrombin-dependent anti-neuropathic activity of exogenous TMα and, most probably, endogenous TM.

We tested whether NNC 55-0396 (NNC), a T-type calcium channel (T-channel) blocker, reduces the brain injury caused by middle cerebral artery occlusion and reperfusion (MCAO/R) in mice. NNC, administered i.c.v. before the occlusion, greatly reduced the MCAO/R-induced brain infarct and neurological dysfunctions, although it, given toward the end of occlusion, was less effective. Systemic administration of NNC before the occlusion also attenuated the infarct and neurological dysfunctions. Our data imply that blood-brain-barrier-permeable T-channel blockers such as NNC are capable of reducing MCAO/R-induced brain damage, and that T-channels are involved in neuronal damage induced by ischemia rather than reperfusion. Keywords: T-type calcium channel, Middle cerebral artery occlusion, Ischemia-reperfusion

Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting adverse reaction in cancer patients treated with several cytotoxic anticancer agents including paclitaxel. Duloxetine, an antidepressant known as a serotonin-noradrenalin reuptake inhibitor, is the only agent that has moderate evidence for the use to treat painful CIPN. The present retrospective cohort study aimed to analyze risk factors for paclitaxel-induced peripheral neuropathy (PIPN), and investigate ongoing prescription drug use for PIPN in Japan. Female breast and gynecologic cancer patients who underwent paclitaxel-based chemotherapy at a single center in Japan between January 2016 and December 2019 were enrolled in this study. Patients’ information obtained from electronic medical records were statistically analyzed to test possible risk factors on PIPN diagnosis. Patients’ age, total paclitaxel dose, the history of female hormone-related diseases, hypertension and body mass index (BMI), but not additional platinum agents, were significantly associated with increased PIPN diagnosis. Drugs prescribed for PIPN included duloxetine, pregabalin, mecobalamin and Goshajinkigan, a polyherbal medicine, regardless of poor evidence for their effectiveness against CIPN, and were greatly different between breast and gynecologic cancer patients diagnosed with PIPN at the departments of Surgery and Gynecology, respectively. Thus, older age, greater total paclitaxel dose, the history of estrogen-related diseases, hypertension and BMI are considered risk factors for PIPN in paclitaxel-based chemotherapy of female cancer patients. It appears an urgent need to establish a guideline of evidence-based pharmacotherapy for PIPN.

We studied the pronociceptive role of proteinase-activated receptor-2 (PAR2) in mouse bladder. In female mice, intravesical infusion of the PAR2-activating peptide, SLIGRL-amide (SL), caused delayed mechanical hypersensitivity in the lower abdomen, namely ‘referred hyperalgesia’, 6–24 h after the administration. The PAR2-triggered referred hyperalgesia was prevented by indomethacin or a selective TRPV1 blocker, and restored by a T-type Ca2+ channel blocker. In human urothelial T24 cells, SL caused delayed prostaglandin E2 production and COX-2 upregulation. Our data suggest that luminal PAR2 stimulation in the bladder causes prostanoid-dependent referred hyperalgesia in mice, which involves the activation of TRPV1 and T-type Ca2+ channels.

We tested whether genetic deletion of Cav3.2 T-type Ca2+ channels abolishes hydrogen sulfide (H2S)-mediated pain signals in mice. In Cav3.2-expressing HEK293 cells, Na2S, an H2S donor, at 100 μM clearly increased Ba2+ currents, as assessed by whole-cell patch-clamp recordings. In wild-type C57BL/6 mice, intraplantar and intracolonic administration of Na2S evoked mechanical allodynia and visceral nociceptive behavior, respectively, which were abolished by TTA-A2, a T-type Ca2+ channel blocker. In Cav3.2-knockout mice of a C57BL/6 background, Na2S caused neither somatic allodynia nor colonic nociception. Our study thus provides definitive evidence for an essential role of Cav3.2 in H2S-dependent somatic and colonic pain. Keywords: Hydrogen sulfide, Cav3.2 T-type calcium channel, Pain

Given the role of macrophage-derived high mobility group box 1 (HMGB1) in chemotherapy-induced peripheral neuropathy (CIPN) caused by paclitaxel, we analyzed the role of HMGB1 and macrophages in the CIPN caused by bortezomib, a proteasome-inhibiting chemotherapeutic agent used for the treatment of multiple myeloma. Repeated administration of bortezomib caused CIPN accompanied by early-stage macrophage accumulation in the dorsal root ganglion. This CIPN was prevented by an anti-HMGB1-neutralizing antibody, thrombomodulin alfa capable of accelerating thrombin-dependent degradation of HMGB1, antagonists of the receptor for advanced glycation end-products (RAGE) and C-X-C motif chemokine receptor 4 (CXCR4), known as HMGB1-targeted membrane receptors, or macrophage depletion with liposomal clodronate, as reported in a CIPN model caused by paclitaxel. In macrophage-like RAW264.7 cells, bortezomib as well as MG132, a well-known proteasome inhibitor, caused HMGB1 release, an effect inhibited by caspase inhibitors but not inhibitors of NF-κB and p38 MAP kinase, known to mediate paclitaxel-induced HMGB1 release from macrophages. Bortezomib increased cleaved products of caspase-8 and caused nuclear fragmentation or condensation in macrophages. Repeated treatment with the caspase inhibitor prevented CIPN caused by bortezomib in mice. Our findings suggest that bortezomib causes caspase-dependent release of HMGB1 from macrophages, leading to the development of CIPN via activation of RAGE and CXCR4.

A neuroimmune crosstalk is involved in somatic and visceral pathological pain including inflammatory and neuropathic components. Apart from microglia essential for spinal and supraspinal pain processing, the interaction of bone marrow-derived infiltrating macrophages and/or tissue-resident macrophages with the primary afferent neurons regulates pain signals in the peripheral tissue. Recent studies have uncovered previously unknown characteristics of tissue-resident macrophages, such as their origins and association with regulation of pain signals. Peripheral nerve macrophages and intestinal resident macrophages, in addition to adult monocyte-derived infiltrating macrophages, secrete a variety of mediators, such as tumor necrosis factor-α, interleukin (IL)-1β, IL-6, high mobility group box 1 and bone morphogenic protein 2 (BMP2), that regulate the excitability of the primary afferents. Neuron-derived mediators including neuropeptides, ATP and macrophage-colony stimulating factor regulate the activity or polarization of diverse macrophages. Thus, macrophages have multitasks in homeostatic conditions and participate in somatic and visceral pathological pain by interacting with neurons.

Given that Cav3.2 T-type Ca2+ channels were functionally regulated by asparagine (N)-linked glycosylation, we examined effects of high glucose on the function of Cav3.2, known to regulate secretory function, in neuroendocrine-like differentiated prostate cancer LNCaP cells. High glucose accelerated the increased channel function and overexpression of Cav3.2 during neuroendocrine differentiation, the former prevented by enzymatic inhibition of N-glycosylation and cleavage of N-glycans. Hyperglycemia thus appears to induce N-linked glycosylation-mediated functional upregulation and overexpression of Cav3.2 in neuroendocrine-like differentiated prostate cancer cells.

We investigated mechanisms for the neuritogenesis caused by prostaglandin E2 (PGE2) or intracellular cyclic AMP (cAMP) in sensory neuron-like ND7/23 cells. PGE2 caused neuritogenesis, an effect abolished by an EP4 receptor antagonist or inhibitors of adenylyl cyclase (AC) or protein kinase A (PKA) and mimicked by the AC activator forskolin, dibutyryl cAMP (db-cAMP), and selective activators of PKA or Epac. ND7/23 cells expressed both Cav3.1 and Cav3.2 T-type Ca2+ channels (T-channels). The neuritogenesis induced by db-cAMP or PGE2 was abolished by T-channel blockers. T-channels were functionally upregulated by db-cAMP. The PGE2/EP4/cAMP/T-channel pathway thus appears to mediate neuritogenesis in sensory neurons.

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.

We examined the effects of intraplantar (i.pl.) administration of NaHS, an H2S donor, known to cause T-type Ca2+ channel (T-channel)-dependent mechanical hyperalgesia, on responsiveness to electric stimulation with 5, 250 and 2000 Hz sine waves (SW) that selectively excites C, Aδ and Aβ fibers, respectively. NaHS, given i.pl., caused behavioral hypersensitivity to SW stimulation at 5 Hz, but not 250 or 2000 Hz, in rats. NaHS also enhanced phosphorylation of spinal ERK following 5 Hz SW stimulation. Three distinct T-channel blockers abolished the NaHS-induced behavioral hypersensitivity to 5 Hz SW stimulation. Thus, H2S selectively sensitizes C-fiber nociceptors via T-channels.

Cystitis-related bladder pain involves RAGE activation by HMGB1, and increased Cav3.2 T-type Ca2+ channel activity by H2S, generated by upregulated cystathionine-γ-lyase (CSE) in mice treated with cyclophosphamide (CPA). We, thus, investigated possible crosstalk between the HMGB1/RAGE and CSE/H2S/Cav3.2 pathways in the bladder pain development. Bladder pain (nociceptive behavior/referred hyperalgesia) and immuno-reactive CSE expression in the bladder were determined in CPA-treated female mice. Cell signaling was analyzed in urothelial T24 and macrophage-like RAW264.7 cells. The CPA-induced bladder pain was abolished by pharmacological inhibition of T-type Ca2+ channels or CSE, and genetic deletion of Cav3.2. The CPA-induced CSE upregulation, as well as bladder pain was prevented by HMGB1 inactivation, inhibition of HMGB1 release from macrophages, antagonists of RAGE or P2X4/P2X7 receptors, and N-acetylcysteine, an antioxidant. Acrolein, a metabolite of CPA, triggered ATP release from T24 cells. Adenosine triphosphate (ATP) stimulated cell migration via P2X7/P2X4, and caused HMGB1 release via P2X7 in RAW264.7 cells, which was dependent on p38MAPK/NF-κB signaling and reactive oxygen species (ROS) accumulation. Together, our data suggest that CPA, once metabolized to acrolein, causes urothelial ATP-mediated, redox-dependent HMGB1 release from macrophages, which in turn causes RAGE-mediated CSE upregulation and subsequent H2S-targeted Cav3.2-dependent nociceptor excitation, resulting in bladder pain.

Cav3.2 T-type Ca2+ channels targeted by H2S, a gasotransmitter, participate in cyclophosphamide-induced cystitis and bladder pain. Given that zinc selectively inhibits Cav3.2 among T-channel isoforms and also exhibits antioxidant activity, we examined whether polaprezinc (zinc-l-carnosine), a medicine for peptic ulcer treatment and zinc supplementation, reveals preventive or therapeutic effects on bladder inflammation and/or pain in the mouse with cyclophosphamide-induced cystitis, a model for interstitial cystitis. Systemic administration of cyclophosphamide caused cystitis-related symptoms including increased bladder weight and vascular permeability, and histological signs of bladder edema, accompanied by bladder pain-like nociceptive behavior/referred hyperalgesia. All these symptoms were significantly attenuated by oral preadministration of polaprezinc at 400 mg/kg. The same dose of polaprezinc also prevented the increased malondialdehyde level, an indicator of lipid peroxidation, and protein upregulation of cystathionine-γ-lyase, an H2S-generating enzyme, but not occludin, a tight junction-related membrane protein, in the bladder tissue of cyclophosphamide-treated mice. Oral posttreatment with polaprezinc at 30–100 mg/kg reversed the nociceptive behavior/referred hyperalgesia in a dose-dependent manner without affecting the increased bladder weight. Together, our data show that zinc supplementation with polaprezinc prevents the cyclophosphamide-induced cystitis probably through the antioxidant activity, and, like T-channel blockers, reverses the established cystitis-related bladder pain in mice, suggesting novel therapeutic usefulness of polaprezinc.

Abstract.: We evaluated the effect of buprenorphine, a mixed agonist for μ-opioid receptors and nociceptin/orphanin FQ peptide (NOP) receptors, in neuropathic rats, using the paw pressure test. Buprenorphine, administered i.p. at 50, but not 20, μg/kg, exhibited naloxone-reversible analgesic activity in naïve rats. In contrast, buprenorphine at 0.5 – 20 μg/kg produced a naloxone-sensitive antihyperalgesic effect in the L5 spinal nerve–injured neuropathic rats. Intrathecal injection of [N-Phe1]nociceptin(1-13)NH2, a NOP-receptor antagonist, reversed the effect of buprenorphine in neuropathic rats, but not in naïve rats. Together, buprenorphine suppresses neuropathic hyperalgesia by activating NOP and opioid receptors, suggesting its therapeutic usefulness in treatment of neuropathic pain. Keywords:: buprenorphine, nociceptin/orphanin FQ peptide receptor, neuropathic pain

Low-voltage-activated T-type Ca2+ channels (T-channels), especially Cav3.2 among the three isoforms (Cav3.1, Cav3.2, and Cav3.3), are now considered to play pivotal roles in processing of pain signals. Cav3.2 T-channels are functionally modulated by extracellular substances such as hydrogen sulfide and ascorbic acid, by intracellular signaling molecules including protein kinases, and by glycosylation. Cav3.2 T-channels are abundantly expressed in both peripheral and central endings of the primary afferent neurons, regulating neuronal excitability and release of excitatory neurotransmitters such as substance P and glutamate, respectively. Functional upregulation of Cav3.2 T-channels is involved in the pathophysiology of inflammatory, neuropathic, and visceral pain. Thus, Cav3.2 T-channels are considered to serve as novel targets for development of drugs for treatment of intractable pain resistant to currently available analgesics. Keywords:: Cav3.2, T-type calcium channel, neuropathy, visceral pain, hydrogen sulfide

We examined if TRPA1, like TRPV1, contributes to pancreatic nociceptor excitation following proteinase-activated receptor-2 (PAR2) stimulation and to pancreatitis-related pain in mice. A PAR2-activating peptide, infused into the pancreatic duct, caused spinal Fos expression, which was prevented by AP18, a TRPA1 inhibitor. Repeated administration of cerulein caused referred hyperalgesia accompanying pancreatitis, which was reversed by SB366791, a TRPV1 inhibitor, but not AP18. AP18, administered in combination with a subeffective dose of SB366791, significantly suppressed the referred hyperalgesia. Our findings suggest that TRPA1, like TRPV1, mediates PAR2-triggered pancreatic nociception and that TRPA1 in collaboration with TRPV1 latently contributes to pancreatitis-related pain. Keywords:: transient receptor potential ankyrin-1 (TRPA1), proteinase-activated receptor-2 (PAR2), pancreatic pain

Luminal hydrogen sulfide (H2S), a gasotransmitter, causes colonic pain / referred hyperalgesia in mice, most probably via activation of T-type Ca2+ channels. Here we analyzed the mechanisms for H2S-induced facilitation of colonic pain signals. Intracolonic administration of NaHS, an H2S donor, evoked visceral pain−like nociceptive behavior and referred hyperalgesia in mice, an effect abolished by NNC 55-0396, a selective T-type Ca2+-channel blocker, or by knockdown of Cav3.2. AP18, a TRPA1 blocker, also prevented the NaHS-induced colonic pain and referred hyperalgesia. These findings demonstrate that H2S-induced colonic pain and referred hyperalgesia require activation of both Cav3.2 and TRPA1 channels in mice. Keywords:: hydrogen sulfide, T-type calcium channel, TRPA1

We performed this study to determine if curcumin affects pro-inflammatory responses to activation of proteinase-activated receptor-2 (PAR2) in human pulmonary adenocarcinoma A549 cells. Curcumin completely inhibited the PAR2-triggered prostaglandin E2 (PGE2) production, but notably not interleukin-8 release. Cyclooxygenase-2 (COX-2) upregulation, but not its upstream activation of mitogen-activated protein kinases, caused by PAR2 stimulation was partially inhibited by curcumin. Curcumin inhibited the PAR2-triggered phosphorylation of I-κB, an indicator for nuclear factor-κB (NF-κB) activation, and also its upstream signal Akt, which is known to contribute to PAR2-triggered PGE2 formation, but not COX-2 upregulation. Collectively, curcumin inhibits the PAR2-triggered PGE2 production by suppressing COX-2 upregulation and Akt/NF-κB signals in A549 cells. Keywords:: curcumin, proteinase-activated receptor-2, prostaglandin E2

Proteinase-activated receptor-2 (PAR2) triggers upregulation of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) formation in human alveolar epithelial A549 cells. This COX-2 upregulation appears to involve the Src / epidermal growth factor (EGF) receptor / p38 MAP kinase (p38MAPK) pathway and also the cAMP-response element–binding protein (CREB) pathway. Here, we investigated the roles of nuclear factor-κB (NF-κB)–related signals in the PAR2-triggered PGE2 release / COX-2 upregulation in A549 cells. The PAR2-triggered PGE2 release was clearly blocked by an inhibitor of the NF-κB pathway. Stimulation of PAR2 actually caused phosphorylation of inhibitor-κB, an indicator of NF-κB activation, an effect being blocked by inhibitors of MEK, phosphatidylinositol 3–kinase (PI3-kinase), and Akt, but little or not by inhibitors of p38MAPK and JNK. Stimulation of PAR2 also caused phosphorylation of Akt, an effect suppressed by inhibitors of PI3-kinase and MEK. Nonetheless, the PAR2-triggered upregulation of COX-2 was resistant to inhibitors of NF-κB, PI3-kinase, and Akt, but was attenuated by inhibitors of MEK and JNK. Stimulation of PAR2 induced phosphorylation of CREB, an effect abolished by an inhibitor of MEK but not inhibitors of p38MAPK and EGF receptor. These findings demonstrate that the MEK / ERK / PI3-kinase / Akt / NF-κB pathway is involved in PAR2-triggered PGE2 formation, but not upregulation of COX-2 that is dependent on activation of ERK/CREB and JNK in addition to p38MAPK. Keywords:: proteinase-activated receptor-2 (PAR2), nuclear factor-κB (NF-κB), prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), human alveolar epithelial cell line A549

We investigated the effects of NaHS, a hydrogen sulfide (H2S) donor, on the tension of isolated mouse and guinea-pig bronchial rings. NaHS at 0.01 – 10 mM had no effect on the tone of those preparations without precontraction. When the preparation was precontracted with carbachol, NaHS at 0.1 – 3 mM strongly relaxed the mouse rings, but produced only slight relaxation in the guinea-pig rings. The NaHS-induced relaxation in the mouse bronchus was resistant to inhibitors of ATP-sensitive K+ channels, soluble guanylyl cyclase, cyclooxygenase (COX)-1 or COX-2, and antagonists of tachykinin receptors. Thus, NaHS evokes bronchodilation in mice, suggesting a possible role for H2S in the respiratory system. Keywords:: hydrogen sulfide (H2S), smooth muscle, bronchodilation

PAR-2 is the second member of the family of proteinase-activated receptors activated by trypsin, tryptase, and several other serine proteinases. In order to evaluate the therapeutic potential for PAR-2, we have performed studies on PAR-2-mediated signal transduction and investigated the effects of PAR-2 gene deficiency in disease models. In addition to the G-protein-coupled receptor-mediated common signal transduction pathways, inositol 1,4,5-trisphosphate production and mobilization of Ca2+, PAR-2 can also activate multiple kinase pathways, ERK, p38MAPK, JNK, and IKK, in a cell-type specific manner. The studies using PAR-2-genedeficient mice highlighted critical roles of PAR-2 in progression of skin and joint inflammation. We also describe the development and evaluation of potent and metabolically stable PAR-2 agonists in multiple assay systems both in vitro and in vivo. The structure-activity relationship analysis indicated the improved potencies of furoylated peptides. Furthermore, the resistance of the furoylated peptide against aminopeptidase contributed to the highly potent and sustained effects of the peptide in vivo. These studies suggest the potential therapeutic importance of PAR-2 in inflammatory diseases. Also, the PAR-2-gene-deficient mice and the potent and metabolically stable agonists are shown to be useful tools for evaluating the potency of PAR-2 as a therapeutic target. Keywords:: proteinase-activated receptor (PAR)-2, mitogen-activated peptide kinase, PAR-2-gene-deficient mice, PAR-2-activating peptide

Protease-activated receptors (PARs) 1 and 2 are expressed in capsaicin-sensitive sensory neurons, being anti- and pro-nociceptive, respectively. Given the possible cross talk between PAR-2 and capsaicin receptors, we investigated if PAR-2 activation could facilitate capsaicin-evoked visceral pain and referred hyperalgesia in the mouse and also examined the effect of PAR-1 activation in this model. Intracolonic (i.col.) administration of capsaicin triggered visceral pain-related nociceptive behavior, followed by referred hyperalgesia. The capsaicin-evoked visceral nociception was suppressed by intraperitoneal (i.p.) TFLLR-NH2, a PAR-1-activating peptide, but not FTLLR-NH2, a control peptide, and unaffected by i.col. TFLLR-NH2. SLIGRL-NH2, a PAR-2-activating peptide, but not LRGILS-NH2, a control peptide, administered i.col., facilitated the capsaicin-evoked visceral nociception 6 – 18 h after administration, while i.p. SLIGRL-NH2 had no effect. The capsaicin-evoked referred hyperalgesia was augmented by i.col. SLIGRL-NH2, but not LRGILS-NH2, 6 – 18 h after administration, and unaffected by i.p. SLIGRL-NH2, and i.p. or i.col. TFLLR-NH2. Our data suggest that PAR-1 is antinociceptive in processing of visceral pain, whereas PAR-2 expressed in the colonic luminal surface, upon activation, produces delayed sensitization of capsaicin receptors, resulting in facilitation of visceral pain and referred hyperalgesia. Keywords:: protease-activated receptor, visceral pain, capsaicin, referred hyperalgesia

We analyzed signaling mechanisms for prostaglandin E2 (PGE2) production following activation of proteinase-activated receptor-1 (PAR1), a thrombin receptor, in preosteoblastic MC3T3-E1 cells. PAR1 stimulation caused PGE2 release, an effect suppressed by inhibitors of COX-1, COX-2, iPLA2, cPLA2, MAP kinases (MAPKs), Src, EGF receptor (EGFR) tyrosine kinase (EGFR-TK) and matrix metalloproteinase (MMP), but not by an intracellular Ca2+ chelator or inhibitors of PI3 kinase, protein kinase C (PKC) and NF-κB. PAR1 activation induced phosphorylation of MAPKs and upregulation of COX-2. The phosphorylation of p38 MAPK was suppressed by inhibitors of Src and EGFR-TK. The COX-2 upregulation was dependent on ERK, p38, EGFR-TK, Src, and COX-2 itself. PAR1 activation also induced MEK-dependent phosphorylation of cAMP response element binding protein (CREB). All inhibitors of EP1, EP2, EP3 and EP4 receptors suppressed the PAR1- triggered PGE2 release. Exogenously applied PGE2 facilitated PAR1-triggered COX-2 upregulation, but it alone had no effect. Together, the PAR1-mediated PGE2 production in MC3T3-E1 cells appears to involve iPLA2 and cPLA2 for arachidonic acid release, and the MEK/ERK/CREB and Src/MMP/EGFR/p38 pathways for COX-2 upregulation, which is facilitated by endogenous PGE2 formed by COX-2. These signaling mechanisms might underlie the role of the thrombin/PAR1/PGE2 system in the early stage of the bone healing. [ABSTRACT FROM AUTHOR]