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Anoctamins (ANOs) are multifunctional membrane proteins that consist of 10 homologs. ANO1 (TMEM16A) and ANO2 (TMEM16B) are anion channels activated by intracellular calcium that meditate numerous physiological functions. ANO6 is a scramblase that redistributes phospholipids across the cell membrane. The other homologs are not well characterized. We found ANO9/TMEM16J is a cation channel activated by a cAMP-dependent protein kinase A (PKA). Intracellular cAMP-activated robust currents in whole cells expressing ANO9, which were inhibited by a PKA blocker. A cholera toxin that persistently stimulated adenylate cyclase activated ANO9 as did the application of PKA. The cAMP-induced ANO9 currents were permeable to cations. The cAMP-dependent ANO9 currents were augmented by intracellular Ca2+. Ano9 transcripts were predominant in the intestines. Human intestinal SW480 cells expressed high levels of Ano9 transcripts and showed PKA inhibitor-reversible cAMP-dependent currents. We conclude that ANO9 is a cation channel activated by a cAMP/PKA pathway and could play a role in intestine function.

Spinal muscular atrophy and hereditary motor and sensory neuropathies are characterized by muscle weakness and atrophy caused by the degenerations of peripheral motor and sensory nerves. Recent advances in genetics have resulted in the identification of missense mutations in TRPV4 in patients with these hereditary neuropathies. Neurodegeneration caused by Ca(2+) overload due to the gain-of-function mutation of TRPV4 was suggested as the molecular mechanism for the neuropathies. Despite the importance of TRPV4 mutations in causing neuropathies, the precise role of TRPV4 in the sensory/motor neurons is unknown. Here, we report that TRPV4 mediates neurotrophic factor-derived neuritogenesis in developing peripheral neurons. TRPV4 was found to be highly expressed in sensory and spinal motor neurons in early development as well as in the adult, and the overexpression or chemical activation of TRPV4 was found to promote neuritogenesis in sensory neurons as well as PC12 cells, whereas its knockdown and pharmacologic inhibition had the opposite effect. More importantly, nerve growth factor or cAMP treatment up-regulated the expression of phospholipase A(2) and TRPV4. Neurotrophic factor-derived neuritogenesis appears to be regulated by the phospholipase A(2)-mediated TRPV4 pathway. These findings show that TRPV4 mediates neurotrophic factor-induced neuritogenesis in developing peripheral nerves. Because neurotrophic factors are essential for the maintenance of peripheral nerves, these findings suggest that aberrant TRPV4 activity may lead to some types of pathology of sensory and motor nerves.

Significance Benign prostatic hyperplasia (BPH) is characterized by an enlargement of the prostate gland, a common disease in elderly men. Excessive testosterone is considered to cause BPH. However, its etiologic mechanisms are elusive. We found that ANO1, a Ca 2+ -activated Cl − channel, is essential for the testosterone-induced BPH. ANO1 was highly expressed in dihydrotestosterone (DHT)-treated prostate epithelial cells. The selective knockdown of ANO1 suppressed DHT-induced cell proliferation. Surprisingly, we found that there were three androgen-response elements in the ANO1 promoter region, which were relevant for the DHT-dependent induction of ANO1. Intraprostate treatment of Ano1 siRNA inhibited the prostate enlargement in vivo. Thus, ANO1 appears essential for the development of prostate hyperplasia and becomes a useful target for treating BPH.

To compare the efficacy of intrathecal methotrexate single therapy with three-drug combination therapy in patients with leptomeningeal carcinomatosis.Fifty-five patients who had pathologically proven leptomeningeal carcinomatosis of a solid tumor were evaluated in terms of pathological response. Group M (n = 29) received methotrexate 15 mg and group MHA (n = 26) received methotrexate 15 mg, hydrocortisone 15 mg/m(2) and ara-C 30 mg/m(2) twice a week intrathecally until a cytological response was obtained.Primary sites of the tumor were the lung (n = 33), breast (n = 13) and stomach (n = 5). The pathology of 45 patients was adenocarcinoma. The cytological response rate to intrathecal chemotherapy was significantly higher in the MHA group than in the M group (38.5 vs 13.8%, P = 0.036). The median survival was 18.6 weeks in the MHA arm and 10.4 weeks in the M arm (P = 0.029).Combination intrathecal chemotherapy with methotrexate, cytosine arabinoside and hydrocortisone showed more favorable effects than methotrexate single therapy for leptomeningeal carcinomatosis in solid tumors.

Anoctamin 1 (ANO1)/TMEM16A is a Cl− channel activated by intracellular Ca2+ mediating numerous physiological functions. However, little is known of the ANO1 activation mechanism by Ca2+. Here, we demonstrate that two helices, “reference” and “Ca2+ sensor” helices in the third intracellular loop face each other with opposite charges. The two helices interact directly in a Ca2+-dependent manner. Positively and negatively charged residues in the two helices are essential for Ca2+-dependent activation because neutralization of these charges change the Ca2+ sensitivity. We now predict that the Ca2+ sensor helix attaches to the reference helix in the resting state, and as intracellular Ca2+ rises, Ca2+ acts on the sensor helix, which repels it from the reference helix. This Ca2+-dependent push-pull conformational change would be a key electromechanical movement for gating the ANO1 channel. Because chemical activation of ANO1 is viewed as an alternative means of rescuing cystic fibrosis, understanding its gating mechanism would be useful in developing novel treatments for cystic fibrosis. Electronic supplementary material The online version of this article (doi:10.1007/s00424-014-1603-2) contains supplementary material, which is available to authorized users.

Capsaicin, a pungent ingredient of hot peppers, causes excitation of small sensory neurons, and thereby produces severe pain. A nonselective cation channel activated by capsaicin has been identified in sensory neurons and a cDNA encoding the channel has been cloned recently. However, an endogenous activator of the receptor has not yet been found. In this study, we show that several products of lipoxygenases directly activate the capsaicin-activated channel in isolated membrane patches of sensory neurons. Among them, 12- and 15-( S )-hydroperoxyeicosatetraenoic acids, 5- and 15-( S )-hydroxyeicosatetraenoic acids, and leukotriene B 4 possessed the highest potency. The eicosanoids also activated the cloned capsaicin receptor (VR1) expressed in HEK cells. Prostaglandins and unsaturated fatty acids failed to activate the channel. These results suggest a novel signaling mechanism underlying the pain sensory transduction.

Intracellular Ca(2+) signal is a key regulator of axonal growth during brain development. As transient receptor potential (TRP) channels are permeable to Ca(2+) and mediate numerous brain functions, it is conceivable that many TRP channels would regulate neuronal differentiation. We therefore screened TRP channels that are involved in the regulation of neurite growth. Among the TRP channels, the Trpm2 level was inversely associated with neurite growth. TRPM2 was highly expressed in embryonic brain. Pharmacological perturbation or knockdown of TRPM2 markedly increased the axonal growth, whereas its overexpression inhibited the axonal growth. Addition of ADP ribose, an endogenous activator of TRPM2, to PC12 cells significantly repressed the axonal growth. TRPM2 was actively involved in the neuronal retraction induced by cerebrospinal fluid-rich lysophosphatidic acid (LPA). More importantly, neurons isolated from the brain of Trpm2-deficient mice have significantly longer neurites with a greater number of spines than those obtained from the brain of wild-type mice. Therefore, we conclude that TRPM2 mediates the LPA-induced suppression of axonal growth, which provides a long-sought mechanism underlying the effect of LPA on neuronal development.

Intracardiac metastasis as the initial presentation of malignant neoplasm is very rare. We report here on a 64-year-old man with non-small cell lung cancer (NSCLC) initially presenting with intracardiac metastasis which was identified with 18-F fluorodeoxyglucose positron emission tomography (FDG PET). The patient was admitted with complaints of exertional dyspnea and vague chest discomfort that had developed a few weeks ago. Two-dimensional echocardiography revealed a heart mass attached to its akinetic wall in the right ventricular chamber. CT and MRI demonstrated a large tumor involving the epicardium and myocardium in the right ventricle, and there was a mass in the right lower lobe of the lung along with multiple lymphadenopathies. Cytologic examination of the percutaneous needle aspiration of a lymph node in the anterior mediastinum revealed malignant epithelial cell nests, and this was strongly suggestive of squamous cell carcinoma. Subsequent FDG PET confirmed that the intracardiac mass had an abnormally increased FDG uptake, and again this was strongly suggestive of malignancy. By systemically considering these imaging studies, we were able to diagnose the mass as intracardiac metastasis of NSCLC.

Jumonji (JMJ) can function as a transcriptional repressor and plays critical roles in embryonic development including heart development in mice. Although JMJ has been suggested to play a role in cell growth, the molecular mechanisms have not been resolved. The present data demonstrate that JMJ interacts with the retinoblastoma protein (Rb), one of the master regulatory genes of cell cycle. JMJ potentiates the repression function of Rb on E2F activities, leading to reduced cell cycle progression. The transcriptional repression domain of JMJ is critical for the interaction with Rb as well as repression of cell cycle. The physiological relevance of the association between Rb and JMJ was assessed in cardiomyocytes. Primary cardiomyocytes cultured from homozygous jmj knock-out mouse embryos (jmj mutants) show increased cell mitosis in a cardiomyocyte-specific manner. Reporter gene analyses demonstrate that promoter activities of cyclin D1, cyclin D2, and Cdc2 are up-regulated in jmj mutant cardiomyocytes. These data suggest that JMJ down-regulates the cell growth via interaction with Rb, which would provide important insights into the cardiac defects observed in jmj mutant mice.

Vanilloid receptor 1 (VR1), a capsaicin receptor, is known to play a major role in mediating inflammatory thermal nociception. Although the physiological role and biophysical properties of VR1 are known, the mechanism of its activation by ligands is poorly understood. Here we show that VR1 must be phosphorylated by Ca2+-calmodulin dependent kinase II (CaMKII) before its activation by capsaicin. In contrast, the dephosphorylation of VR1 by calcineurin leads to a desensitization of the receptor. Moreover, point mutations in VR1 at two putative consensus sites for CaMKII failed to elicit capsaicin-sensitive currents and caused a concomitant reduction in VR1 phosphorylation in vivo. Such mutants also lost their high affinity binding with [3H]resiniferatoxin, a potent capsaicin receptor agonist. We conclude that the dynamic balance between the phosphorylation and dephosphorylation of the VR1 channel by CaMKII and calcineurin, respectively, controls the activation/desensitization states by regulating VR1 binding. Furthermore, because sensitization by protein kinase A and C converge at these sites, phosphorylation stress in the cell appears to control a wide range of excitabilities in response to various adverse stimuli.

This study was conducted to investigate the efficacy of the induction chemotherapy followed by radiotherapy in anal cancer. Twenty-three patients diagnosed with anal cancer between March 1991 and February 1999 were treated with induction chemotherapy with 5-fluorouracil based regimens followed by external beam radiation. After a median follow-up of 78 months, the overall survival and the disease-free survival at 5 years was 71.3 and 67.5%, respectively. The colostomy-free survival at 5 years was 91%, as the inguinal lymph nodes were the most frequent site of relapse. Serious side effects did not occur and late complications did not develop either. Induction chemotherapy followed by radiotherapy in patients with anal cancer is effective in terms of its response and preserving the anal sphincter without serious acute and late complications.

The capsaicin-sensitive vanilloid receptor (VR1) was recently shown to play an important role in inflammatory pain (hyperalgesia), but the underlying mechanism is unknown. We hypothesized that pain-producing inflammatory mediators activate capsaicin receptors by inducing the production of fatty acid agonists of VR1. This study demonstrates that bradykinin, acting at B2 bradykinin receptors, excites sensory nerve endings by activating capsaicin receptors via production of 12-lipoxygenase metabolites of arachidonic acid . This finding identifies a mechanism that might be targeted in the development of new therapeutic strategies for the treatment of inflammatory pain.

The association between aplastic crisis and human parvovirus (HPV) B19 infection is well described in patients with sickle cell anemia. This association has also been described, although much less frequently, in patients with hereditary spherocytosis (HS). However, most cases of aplastic crises in patients with HS and induced by HPV B19 have been reported in children or adolescents. In this paper, we describe an aplastic crisis induced by HPV B19 in an adult with HS. A 34-year-old female presented with presyncope, febrile sensation, and myalgia. The complete blood counts showed severe anemia. The peripheral blood smear revealed spherocytosis with reticulocytopenia and pancytopenia. The direct Coombs' test was negative; the osmotic fragility test was positive. In the bone marrow aspirates, a few giant pronormoblasts with deep blue cytoplasm, pseudopods, and intracellular inclusion bodies were observed. The patient was given eight units of packed red blood cells. HPV B19 infection was proven by the presence of IgM antibodies to HPV B19 and the detection of viral DNA using the PCR technique. To the best of our knowledge, this is the first report in Korea that describes an adult with aplastic crisis presenting initially with HS.

Ca(2+)-activated Cl(-) channels (CaCCs) are a class of Cl(-) channels activated by intracellular Ca(2+) that are known to mediate numerous physiological functions. In 2008, the molecular identity of CaCCs was found to be anoctamin 1 (ANO1/TMEM16A). Its roles have been studied in electrophysiological, histological, and genetic aspects. ANO1 is known to mediate Cl(-) secretion in secretory epithelia such as airways, salivary glands, intestines, renal tubules, and sweat glands. ANO1 is a heat sensor activated by noxious heat in somatosensory neurons and mediates acute pain sensation as well as chronic pain. ANO1 is also observed in vascular as well as airway smooth muscles, controlling vascular tone as well as airway hypersensitivity. ANO1 is upregulated in numerous types of cancers and thus thought to be involved in tumorigenesis. ANO1 is also found in proliferating cells. In addition to ANO1, involvement of its paralogs in pathophysiological conditions was also reported. ANO2 is involved in olfaction, whereas ANO6 works as a scramblase whose mutation causes a rare bleeding disorder, the Scott syndrome. ANO5 is associated with muscle and bone diseases. Recently, an X-ray crystal structure of a fungal TMEM16 was reported, which explains a precise molecular gating mechanism as well as ion conduction or phospholipid transport across the plasma membrane.