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This study was a comparative analysis of the artificial degradation of silk by Xenon arc lamp, long-wavelength ultraviolet(UV-A), and dry heat. Two types of silk specimens with different thicknesses were treated under each condition for 24, 72, 120, and 168 hours. Tensile strength, yellowing index, and fiber surface changes were analyzed. Deterioration behavior after treatment was examined. The factor that most rapidly promoted degradation was UV-A exposure. The Xenon arc lamp produced a gentle decrease in tensile strength and a low yellowness index. Heat produced little change in the physical properties of the silk specimens. The comparative data indicated that silk degradation upon exposure to Xenon arc lamp was slow, with a low yellowness index. Thus, Xenon arc lamp is acceptable for the conservation of textile cultural heritage, along with reinforcement by sewing.

This study aimed to evaluate whether fluvoxamine reduces clinical deterioration in adult patients with mild to moderate coronavirus disease 2019 (COVID-19), and to identify risk factors for clinical deterioration in patients admitted to a community treatment center (CTC).A randomized, placebo-controlled trial was conducted in a CTC, in Seoul, Korea from January 15, 2021, to February 19, 2021. Symptomatic adult patients with positive results of severe acute respiratory syndrome coronavirus 2 real time-polymerase chain reaction within 3 days of randomization were assigned at random to receive 100 mg of fluvoxamine or placebo twice daily for 10 days. The primary outcome was clinical deterioration defined by any of the following criteria: oxygen requirement to keep oxygen saturation over 94.0%, aggravation of pneumonia with dyspnea, or World Health Organization clinical progression scale 4 or greater.Of 52 randomized participants [median (interquartile range) age, 53.5 (43.3 - 60.0) years; 31 (60.0%) men], 44 (85.0%) completed the trial. Clinical deterioration occurred in 2 of 26 patients in each group (In this study of adult patients with symptomatic COVID-19 who were admitted to the CTC, there was no significant differences in clinical deterioration between patients treated with fluvoxamine and placebo (ClinicalTrials.gov Identifier: NCT04711863).

Sarcopenia is a disease of old age characterized by decreased muscle mass and strength. Branched-chain amino acids (BCAAs) promote muscle mass synthesis and increase muscle strength. We aimed to develop a dietary amino acid database and to examine the association between BCAA intake and handgrip strength in Korean older adults. Data from the Korea National Health and Nutrition Examination Survey 2014–2018 were used. Overall, 4852 participants aged ≥65 years were included in the study. Demographic, lifestyle, and health data were obtained through interviews and questionnaires. The 24-h recall method was used to assess dietary intake. An amino acid database was established using the 9th revision of the Korean Standard Food Composition Table. The mean handgrip strength was estimated from triplicate measurements obtained using the dominant hand. Multivariable linear regression analysis was performed to assess the association between BCAA intake and handgrip strength. Grains greatly contributed to amino acid intake, however, a significant negative association was observed between handgrip strength and increased BCAA intake through grains. In the fully adjusted model, leucine intake and handgrip strength showed a positive association. Thus, consuming BCAAs (especially leucine) via a variety of food sources can help maintain muscle health in older people.

The development of resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) occurs by various mechanisms and appears to be almost inevitable, even in patients with lung cancer who initially respond well to EGFR-TKIs. Consequently, considerable efforts have been made to develop more effective EGFR-TKIs. Therefore, an understanding of the mechanisms behind TKI resistance is essential for improving EGFR-TKI therapeutic efficacy in non-small cell lung cancer (NSCLC) patients. In this study, we discovered that overexpression of antioxidant-responsive element (ARE)-containing Nrf2 target genes by increased transactivation of Nrf2 occurred because of an acquired Keap1 mutation in the gefitinib-resistant (GR) NSCLC cell line we established. These GR cells also acquired cross-resistance to the irreversible EGFR-TKIs, afatinib and osimertinib, and showed increased viability, invasiveness, proliferation, and tumorigenicity both in vitro and in vivo. These results were confirmed by the fact that inhibition of Nrf2 activity, either by treatment with brusatol or by inducing expression of exogenously introduced wild-type Keap1, suppressed tumor cell proliferation and tumorigenicity in vitro and in vivo. Our data suggest that disruption of the Keap1-Nrf2 pathway is one of the mechanisms by which EGFR-TKI resistance occurs, a fact that must be considered when treating patients with EGFR-TKI.-Park, S.-H., Kim, J. H., Ko, E., Kim, J.-Y., Park, M.-J., Kim, M. J., Seo, H., Li, S., Lee, J.-Y. Resistance to gefitinib and cross-resistance to irreversible EGFR-TKIs mediated by disruption of the Keap1-Nrf2 pathway in human lung cancer cells.

Streptococcus pneumoniae is an important human pathogen responsible for more than 2 million deaths annually worldwide. The airway epithelium acts as the first-line of defense against pneumococcal infections by regulating acute inflammation against invading pneumococcus. Despite the intact adaptive immunity, failure in early defense due to loss of pattern recognition receptors (PRRs) and/or acute phase proteins (APPs) results in detrimental damage and death. C-reactive protein (CRP), the first found APP, is a member of the pentraxin family of proteins and an important soluble PRR for pneumococcus. CRP and another short pentraxin, serum amyloid P, are critical for acute defense against pneumococcal infection. However, the role of the long pentraxin PTX3 in regulating pneumococcal infections is unknown. In this study, PTX3 expression was upregulated by pneumococcus in epithelial cells and in lungs of mice. In addition, PTX3 potentiated pneumococcal inflammation; overexpression of PTX3 enhanced pneumococcus-induced cytokine expression, whereas knock-down of PTX3 with siPTX3 inhibited the cytokine expression. Furthermore, PTX3 deficiency indeed ameliorated acute inflammation and protected mice against death following pneumococcal infection. Pneumococcal toxin pneumolysin was responsible for PTX3 expression and upregulated PTX3 expression via JNK MAPK signaling. These data implicate PTX3 as a novel therapeutic target for the control of acute inflammation by pneumococcus.

Objective This study aimed to examine the cross-sectional association between adherence to dietary guidelines for type 2 diabetes mellitus (T2DM) and depression. Methods A total of 3457 patients with T2DM were included. Dietary information was obtained using 24-h recall, and adherence to dietary guidelines was estimated using the Korean Diabetes Association-Korean Ministry of Health and Welfare index. Depression was evaluated using a questionnaire. Participants who had responded “yes” to any of the three questions related to diagnosis, current condition, and treatment were defined as depression. Multivariable logistic regression analysis was used to examine the association between adherence to dietary guidelines and depression. Results T2DM patients with a lower score on adherence to dietary guidelines were more likely to report depression than those with a higher score (Odds ratios (OR): 0.51, 95% Confidence Interval (CI): 0.30–0.87). Patients with poor adherence to moderate calorie consumption (OR: 1.65, 95% CI: 1.07–2.52) and regular meal patterns (OR: 1.69, 95% CI: 1.15–2.50) were more likely to report depression. However, patients with poor adherence to low sodium intake were less likely to report depression (OR: 0.60, 95% CI: 0.40–0.88), and no association was observed between moderate carbohydrate consumption, sufficient vegetable/seaweed consumption, and moderate alcohol consumption and depression. Conclusion Good adherence to dietary guidelines was closely associated with a lower prevalence of depression among individuals with type 2 diabetes, but the specific guidelines were inconsistent. Systematic and continuous nutrition education for individuals with T2DM is necessary to improve their mental health outcomes.

All forms of cell signaling occur in discreet cellular microdomains in which the ER is the main participant and include microdomains formed by the ER with lysosomes, endosomes, the nucleus, mitochondria and the plasma membrane. In the microdomains the two opposing organelles transfer and exchange constituents including lipids and ions. As is the case for other forms of signaling pathways, many components of the receptor-evoked Ca 2+ signal are clustered at the ER/PM microdomain, including the Orai1–STIM1 complex. This review discusses recent advances in understanding the molecular components that tether the ER and plasma membrane to form the ER/PM microdomains in which PI(4,5)P 2 is enriched, and how dynamic targeting of the Orai1–STIM1 complex to PI(4,5)P 2 -poor and PI(4,5)P 2 -rich microdomains controls the activity of Orai1 and its regulation by Ca 2+ that is mediated by SARAF.

Tenofovir disoproxil fumarate (TDF), a prodrug of tenofovir, has oral bioavailability (25%) limited by intestinal transport (P-glycoprotein), and intestinal degradation (carboxylesterase). However, the influence of luminal pancreatic enzymes is not fully understood. Physiologically based pharmacokinetic (PBPK) modeling has utility for estimating drug exposure from in vitro data. This study aimed to develop a PBPK model that included luminal enzyme activity to inform dose reduction strategies. TDF and tenofovir stability in porcine pancrelipase concentrations was assessed (0, 0.48, 4.8, 48, and 480 U/ml of lipase; 1 mM TDF; 37°C; 0 to 30 min). Samples were analyzed using mass spectrometry. TDF stability and permeation data allowed calculation of absorption rates within a human PBPK model to predict plasma exposure following 6 days of once-daily dosing with 300 mg of TDF. Regional absorption of drug was simulated across gut segments. TDF was degraded by pancrelipase (half-lives of 0.07 and 0.62 h using 480 and 48 U/ml, respectively). Previously reported maximum concentration ( C max ; 335 ng/ml), time to C max ( T max ; 2.4 h), area under the concentration-time curve from 0 to 24 h (AUC 0–24 ; 3,045 ng · h/ml), and concentration at 24 h ( C 24 ; 48.3 ng/ml) were all within a 0.5-fold difference from the simulated C max (238 ng/ml), T max (3 h), AUC 0–24 (3,036 ng · h/ml), and C 24 (42.7 ng/ml). Simulated TDF absorption was higher in duodenum and jejunum than in ileum (p

The Ca2+ and cAMP/PKA pathways are the primary signaling systems in secretory epithelia that control virtually all secretory gland functions. Interaction and crosstalk in Ca2+ and cAMP signaling occur at multiple levels to control and tune the activity of each other. Physiologically, Ca2+ and cAMP signaling operate at 5–10% of maximal strength, but synergize to generate the maximal response. Although synergistic action of the Ca2+ and cAMP signaling is the common mode of signaling and has been known for many years, we know very little of the molecular mechanism and mediators of the synergism. In this review, we discuss crosstalk between the Ca2+ and cAMP signaling and the function of IRBIT (IP3 receptors binding protein release with IP3) as a third messenger that mediates the synergistic action of the Ca2+ and cAMP signaling.

Objective— Globotriaosylceramide (Gb3) induces K Ca 3.1 downregulation in Fabry disease (FD). We investigated whether Gb3 induces K Ca 3.1 endocytosis and degradation. Approach and Results— K Ca 3.1, especially plasma membrane–localized K Ca 3.1, was downregulated in both Gb3-treated mouse aortic endothelial cells (MAECs) and human umbilical vein endothelial cells. Gb3-induced K Ca 3.1 downregulation was prevented by lysosomal inhibitors but not by a proteosomal inhibitor. Endoplasmic reticulum stress–inducing agents did not induce K Ca 3.1 downregulation. Gb3 upregulated the protein levels of early endosome antigen 1 and lysosomal-associated membrane protein 2 in MAECs. Compared with MAECs from age-matched wild-type mice, those from aged α-galactosidase A (Gla)-knockout mice, an animal model of FD, showed downregulated K Ca 3.1 expression and upregulated early endosome antigen 1 and lysosomal-associated membrane protein 2 expression. In contrast, no significant difference was found in early endosome antigen 1 and lysosomal-associated membrane protein 2 expression between young Gla-knockout and wild-type MAECs. In aged Gla-knockout MAECs, clathrin was translocated close to the cell border and clathrin knockdown recovered K Ca 3.1 expression. Rab5, an effector of early endosome antigen 1, was upregulated, and Rab5 knockdown restored K Ca 3.1 expression, the current, and endothelium-dependent relaxation. Conclusions —Gb3 accelerates the endocytosis and lysosomal degradation of endothelial K Ca 3.1 via a clathrin-dependent process, leading to endothelial dysfunction in FD.

Background & Aims The cyclic adenosine monophosphate (cAMP) and Ca 2+ signaling pathways synergize to regulate many physiological functions. However, little is known about the mechanisms by which these pathways interact. We investigated the synergy between these signaling pathways in mouse pancreatic and salivary gland ducts. Methods We created mice with disruptions in genes encoding the solute carrier family 26, member 6 (Slc26a6 −/− mice) and inositol 1,4,5-triphosphate (InsP 3 ) receptor-binding protein released with InsP 3 (Irbit −/− ) mice. We investigated fluid secretion by sealed pancreatic ducts and the function of Slc26a6 and the cystic fibrosis transmembrane conductance regulator (CFTR) in HeLa cells and in ducts isolated from mouse pancreatic and salivary glands. Slc26a6 activity was assayed by measuring intracellular pH, and CFTR activity was assayed by measuring Cl – current. Protein interactions were determined by immunoprecipitation analyses. Results Irbit mediated the synergistic activation of CFTR and Slc26a6 by Ca 2+ and cAMP. In resting cells, Irbit was sequestered by InsP 3 receptors (IP 3 Rs) in the endoplasmic reticulum. Stimulation of Gs-coupled receptors led to phosphorylation of IP 3 Rs, which increased their affinity for InsP 3 and reduced their affinity for Irbit. Subsequent weak stimulation of Gq-coupled receptors, which led to production of low levels of IP 3 , caused dissociation of Irbit from IP 3 Rs and allowed translocation of Irbit to CFTR and Slc26a6 in the plasma membrane. These processes stimulated epithelial secretion of electrolytes and fluid. These pathways were not observed in pancreatic and salivary glands from Irbit −/− or Slc26a6 −/− mice, or in salivary gland ducts expressing mutant forms of IP 3 Rs that could not undergo protein kinase A–mediated phosphorylation. Conclusions Irbit promotes synergy between the Ca 2+ and cAMP signaling pathways in cultured cells and in pancreatic and salivary ducts from mice. Defects in this pathway could be involved in cystic fibrosis, pancreatitis, or Sjogren syndrome.

Atlastins are large, membrane-bound GTPases that participate in the fusion of endoplasmic reticulum (ER) tubules to generate the polygonal ER network in eukaryotes. They also regulate lipid droplet size and inhibit bone morphogenetic protein (BMP) signaling, though mechanisms remain unclear. Humans have three atlastins (ATL1, ATL2, and ATL3), and ATL1 and ATL3 are mutated in autosomal dominant hereditary spastic paraplegia and hereditary sensory neuropathies. Cellular investigations of atlastin orthologs in most yeast, plants, flies and worms are facilitated by the presence of a single or predominant isoform, but loss-of-function studies in mammalian cells are complicated by multiple, broadly-expressed paralogs. We have generated mouse NIH-3T3 cells lacking all three mammalian atlastins (Atl1/2/3) using CRISPR/Cas9-mediated gene knockout (KO). ER morphology is markedly disrupted in these triple KO cells, with prominent impairment in formation of three-way ER tubule junctions. This phenotype can be rescued by expression of distant orthologs from Saccharomyces cerevisiae (Sey1p) and Arabidopsis (ROOT HAIR DEFECTIVE3) as well as any one of the three human atlastins. Minimal, if any, changes are observed in the morphology of mitochondria and the Golgi apparatus. Alterations in BMP signaling and increased sensitivity to ER stress are also noted, though effects appear more modest. Finally, atlastins appear required for the proper differentiation of NIH-3T3 cells into an adipocyte-like phenotype. These findings have important implications for the pathogenesis of hereditary spastic paraplegias and sensory neuropathies associated with atlastin mutations.

The Ca(2+) second messenger is initiated at ER/PM junctions and propagates into the cell interior to convey the receptor information. The signal is maintained by Ca(2+) influx across the plasma membrane through the Orai and TRPC channels. These Ca(2+) influx channels form complexes at ER/PM junctions with the ER Ca(2+) sensor STIM1, which activates the channels. The function of STIM1 is modulated by other STIM isoforms like STIM1L, STIM2 and STIM2.1/STIM2β and by SARAF, which mediates the Ca(2+)-dependent inhibition of Orai channels. The ER/PM junctions are formed at membrane contact sites by tethering proteins that generate several types of ER/PM junctions, such as PI(4,5)P2-poor and PI(4,5)P2-rich domains. This chapter discusses several properties of the TRPC channels, the Orai channels and the STIMs, their key interacting proteins and how interaction of the STIMs with the channels gates their activity. The chapter closes by highlighting open questions and potential future directions in this field.

The intermediate conductance calcium-activated potassium channel (KCa3.1) mediates proliferation of many cell types including fibroblasts, and is a molecular target for intervention in various cell proliferative diseases. Our previous study showed that reduction of KCa3.1 channel expression by lyso-globotriaosylceramide (lyso-Gb3) inhibits differentiation into myofibroblasts and collagen synthesis, which might lead to development of ascending thoracic aortic aneurysm secondary to Fabry disease. However, how lyso-Gb3 downregulates KCa3.1 channel expression is unknown. Therefore, we aimed to investigate the underlying mechanisms of lyso-Gb3-mediated KCa3.1 channel downregulation, focusing on the cAMP signaling pathway. We found that lyso-Gb3 increased the intracellular cAMP concentration by upregulation of adenylyl cyclase 6 and inhibited ERK 1/2 phosphorylation through the protein kinase A (PKA) pathway, leading to the inhibition of KCa3.1 channel synthesis, not the exchange protein directly activated by cAMP (Epac) pathway. Moreover, lyso-Gb3 suppressed expression of class II phosphatidylinositol 3-kinase C2β (PI3KC2β) by PKA activation, which reduces the production of phosphatidylinositol 3-phosphate [PI(3)P], and the reduced membrane surface expression of KCa3.1 channel was recovered by increasing the intracellular levels of PI(3)P. Consequently, our findings that lyso-Gb3 inhibited both KCa3.1 channel synthesis and surface expression by increasing intracellular cAMP, and controlled surface expression through changes in PI3KC2β-mediated PI(3)P production, suggest that modulation of PKA and PI3KC2β activity to control of KCa3.1 channel expression can be an alternative important target to attenuate ascending thoracic aortic aneurysms in Fabry disease.

Membrane contact sites (MCS) are critical junctions that form between the endoplasmic reticulum (ER) and membranes of various organelles, including the plasma membrane (PM). Signaling complexes, including mediators of Ca2+signaling, are assembled within MCS, such as the ER/PM junction. This is most evident in polarized epithelial cells, such as pancreatic cells. Core Ca2+signaling proteins cluster at the apical pole, the site of inositol 1,4,5-trisphosphate-mediated Ca2+release and Orai1/transient receptor potential canonical-mediated store-dependent Ca2+entry. Recent advances have characterized the proteins that tether the membranes at MCS and the role of these proteins in modulating physiological and pathological intracellular signaling. This review discusses recent advances in the characterization of Ca2+signaling at ER/PM junctions and the relation of these junctions to physiological and pathological Ca2+signaling in pancreatic acini.

The Ca(2+) second messenger is initiated at ER/PM junctions and propagates into the cell interior to convey the receptor information. The signal is maintained by Ca(2+) influx across the plasma membrane through the Orai and TRPC channels. These Ca(2+) influx channels form complexes at ER/PM junctions with the ER Ca(2+) sensor STIM1, which activates the channels. The function of STIM1 is modulated by other STIM isoforms like STIM1L, STIM2 and STIM2.1/STIM2β and by SARAF, which mediates the Ca(2+)-dependent inhibition of Orai channels. The ER/PM junctions are formed at membrane contact sites by tethering proteins that generate several types of ER/PM junctions, such as PI(4,5)P2-poor and PI(4,5)P2-rich domains. This chapter discusses several properties of the TRPC channels, the Orai channels and the STIMs, their key interacting proteins and how interaction of the STIMs with the channels gates their activity. The chapter closes by highlighting open questions and potential future directions in this field.

Fluid and electrolyte homeostasis is a fundamental physiological function required for survival and is associated with a plethora of diseases when aberrant. Systemic fluid and electrolyte composition is regulated by the kidney, and all secretory epithelia generate biological fluids with defined electrolyte composition by vectorial transport of ions and the obligatory water. A major regulatory pathway that immerged in the last several years is regulation of ion transporters by the WNK/SPAK kinases and IRBIT/PP1 pathways. The IRBIT/PP1 pathway functions to reverse the effects of the WNK/SPAK kinases pathway, as was demonstrated for NBCe1-B and CFTR. Since many transporters involved in fluid and electrolyte homeostasis are affected by PP1 and/or calcineurin, it is possible that WNK/SPAK and IRBIT/PP1 form a common regulatory pathway to tune the activity of fluid and electrolyte transport in response to physiological demands.

Bazedoxifene (BZA) acetate, a novel estrogen receptor modulator being developed for the prevention and treatment of postmenopausal osteoporosis, undergoes extensive metabolism in women after oral administration. In this study, the in vitro metabolism of [(14)C]BZA was determined in human hepatocytes and hepatic and intestinal microsomes, and the UDP glucuronosyltransferase (UGT) isozymes involved in the glucuronidation of BZA were identified. In addition, BZA was evaluated for its potential as a substrate of P-glycoprotein (P-gp) transporter in Caco-2 cell monolayers. BZA was metabolized to two monoglucuronides, BZA-4'-glucuronide and BZA-5-glucuronide, in hepatocytes and in liver and intestinal microsomes including jejunum, duodenum, and ileum. Both BZA-4'-glucuronide and BZA-5-glucuronide were major metabolites in the intestinal microsomes, whereas BZA-4'-glucuronide was the predominant metabolite in liver microsomes and hepatocytes. The kinetic parameters of BZA-4'-glucuronide formation were determined in liver, duodenum, and jejunum microsomes and with UGT1A1, 1A8, and 1A10, the most active UGT isoforms involved in the glucuronidation of BZA, whereas those of BZA-5-glucuronide were determined with all the enzyme systems except in liver microsomes and in UGT1A1 because the formation of the BZA-5-glucuronide was too low. K(m) values in liver, duodenum, and jejunum microsomes and UGT1A1, 1A8, and 1A10, were similar and ranged from 5.1 to 33.1 microM for BZA-4'-glucuronide formation and from 2.5 to 11.1 microM for BZA-5-glucuronide formation. V(max) values ranged from 0.8 to 2.9 nmol/(min . mg) protein for BZA-4'-glucuronide and from 0.1 to 1.2 nmol/(min . mg) protein for BZA-5-glucuronide. In Caco-2 cells, BZA appeared to be a P-gp substrate.

We examined the mechanism through which lysophosphatidylcholine (LPC) induces endothelial nitric oxide (eNOS) downregulation. Human umbilical vein endothelial cells (HUVECs) were treated with LPC (50-150 microM) for 0.5-2 h or the reactive oxygen species (ROS) donors, xanthine/xanthine oxidase (X/XO), 1,4-hydroquinone (HQ) or tert-butylhydroperoxide (TBHP) for 2 h. Protein levels of eNOS, superoxide dismutase1 (SOD1), catalase, and phospho-extracellular signal regulated kinase 1/2 (pERK 1/2) were assessed using immunoblotting. LPC treatment reduced SOD1 levels but increased catalase levels. The superoxide donors X/XO and HQ showed similar effects. The hydroperoxide donor TBHP increased SOD1 levels but did not change catalase levels. LPC concentration- and time-dependently decreased eNOS levels, but this effect was blocked by antioxidants and SOD and potentiated by the SOD1 inhibitor, ammonium tetrathiomolybdate. LPC and X/XO inhibited ERK1/2 phosphorylation, whereas TBHP stimulated phosphorylation. Taken together, these data indicate that LPC induces superoxide overload in HUVECs via SOD1 inhibition and downregulates phospho-ERK1/2 and eNOS levels.

Efflux transporters such as P-glycoprotein (P-gp) and multidrug resistance-associated proteins (Mrps) and their contributions to saquinavir (SQV) brain uptake were characterized. Cerebral flow rate was estimated from diazepam uptake and brain vascular volume was assessed using inulin. Mice brains were perfused with buffer containing SQV alone or coperfused with different concentrations of GF120918, a P-gp inhibitor or MK571, a specific Mrp family inhibitor. Inulin, a nonabsorbable marker, was also coperfused in all studies to assess whether the inhibitors altered the physical integrity of the blood-brain barrier (BBB). The estimated cerebral flow rate using diazepam was 250 ml.100g(-1.)min(-1). The brain vascular volume, estimated using inulin, was almost constant (0.94 +/- 0.03 ml.100 g(-1), n = 12) during the perfusion study. SQV uptake kinetics was linear during the sampling period. Inclusion of 10 muM GF120918 in the perfusate resulted in a more than 7-fold increase in the brain distributional volume (i.e., uptake) of SQV. Inclusion of 100 muM MK571 in the perfusate increased SQV apparent brain uptake by more than 4.4-fold, suggesting, for the first time, that Mrp transporters may play an important role in the brain uptake and retention of SQV. Neither GF120918 nor MK571 altered the integrity of the BBB during the time course of the study. Although the current results reaffirm that SQV is a P-gp substrate, this is the first report implicating the Mrp transporter family in the limited brain uptake and retention of SQV in vivo in mice.

The Ca(2+) and cAMP/PKA pathways are the primary signaling systems in secretory epithelia that control virtually all secretory gland functions. Interaction and crosstalk in Ca(2+) and cAMP signaling occur at multiple levels to control and tune the activity of each other. Physiologically, Ca(2+) and cAMP signaling operate at 5-10% of maximal strength, but synergize to generate the maximal response. Although synergistic action of the Ca(2+) and cAMP signaling is the common mode of signaling and has been known for many years, we know very little of the molecular mechanism and mediators of the synergism. In this review, we discuss crosstalk between the Ca(2+) and cAMP signaling and the function of IRBIT (IP3 receptors binding protein release with IP3) as a third messenger that mediates the synergistic action of the Ca(2+) and cAMP signaling.

A central function of epithelia is the control of the volume and electrolyte composition of bodily fluids through vectorial transport of electrolytes and the obligatory H2O. In exocrine glands, fluid and electrolyte secretion is carried out by both acinar and duct cells, with the portion of fluid secreted by each cell type varying among glands. All acinar cells secrete isotonic, plasma-like fluid, while the duct determines the final electrolyte composition of the fluid by absorbing most of the Cl(-) and secreting HCO3 (-). The key transporters mediating acinar fluid and electrolyte secretion are the basolateral Na(+)/K(+) /2Cl(-) cotransporter, the luminal Ca(2+)-activated Cl(-) channel ANO1 and basolateral and luminal Ca(2+)-activated K(+) channels. Ductal fluid and HCO3 (-) secretion are mediated by the basolateral membrane Na(+)-HCO3 (-) cotransporter NBCe1-B and the luminal membrane Cl(-)/HCO3 (-) exchanger slc26a6 and the Cl(-) channel CFTR. The function of the transporters is regulated by multiple inputs, which in the duct include major regulation by the WNK/SPAK pathway that inhibit secretion and the IRBIT/PP1 pathway that antagonize the effects of the WNK/SPAK pathway to both stimulate and coordinate the secretion. The function of these regulatory pathways in secretory glands acinar cells is yet to be examined. An important concept in biology is synergism among signaling pathways to generate the final physiological response that ensures regulation with high fidelity and guards against cell toxicity. While synergism is observed in all epithelial functions, the molecular mechanism mediating the synergism is not known. Recent work reveals a central role for IRBIT as a third messenger that integrates and synergizes the function of the Ca(2+) and cAMP signaling pathways in activation of epithelial fluid and electrolyte secretion. These concepts are discussed in this review using secretion by the pancreatic and salivary gland ducts as model systems.

Modafinil has been used as a psychostimulant for the treatment of narcolepsy. However, its primary mechanism of action remains elusive. Therefore, we examined the effects of modafinil on K(Ca)3.1 channels and vascular smooth muscle contraction. K(Ca)3.1 currents and channel activity were measured using a voltage-clamp technique and inside-out patches in mouse embryonic fibroblast cell line, NIH-3T3 fibroblasts. Intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration was measured, and the phosphorylation of K(Ca)3.1 channel protein was examined using western blotting in NIH-3T3 fibroblasts and/or primary cultured mouse aortic smooth muscle cells (SMCs). Muscle contractions were recorded from mouse aorta and rat pulmonary artery by using a myograph developed in-house. Modafinil was found to inhibit K(Ca)3.1 currents in a concentration-dependent manner, and the half-maximal inhibition (IC(50)) of modafinil for the current inhibition was 6.8 ± 0.7 nM. The protein kinase A (PKA) activator forskolin also inhibited K(Ca)3.1 currents. The inhibitory effects of modafinil and forskolin on K(Ca)3.1 currents were blocked by the PKA inhibitors PKI(14-22) or H-89. In addition, modafinil relaxed blood vessels (mouse aorta and rat pulmonary artery) in a concentration-dependent manner. Modafinil increased cAMP concentrations in NIH-3T3 fibroblasts or primary cultured mouse aortic SMCs and phosphorylated K(Ca)3.1 channel protein in NIH-3T3 fibroblasts. However, open probability and single-channel current amplitudes of K(Ca)3.1 channels were not changed by modafinil. From these results, we conclude that modafinil inhibits K(Ca)3.1 channels and vascular smooth muscle contraction by cAMP-dependent phosphorylation, suggesting that modafinil can be used as a cAMP-dependent K(Ca)3.1 channel blocker and vasodilator.

Aims Excessive endothelial globotriaosylceramide (Gb3) accumulation is associated with endothelial dysfunction and impaired endothelium-dependent relaxation in Fabry disease. In endothelial cells, KCa3.1 channels contribute to endothelium-dependent relaxation. However, the effect of Gb3 on KCa3.1 channels and the underlying mechanisms of Gb3-induced dysfunction are unknown. Herein, we hypothesized that Gb3 accumulation induces KCa3.1 channel dysfunction and aimed to clarify the underlying mechanisms. Methods and results The animal model of Fabry disease, α-galactosidase A ( Gla ) knockout mice, displayed age-dependent KCa3.1 channel dysfunction. KCa3.1 current and the channel expression were significantly reduced in mouse aortic endothelial cells (MAECs) of aged Gla knockout mice, whereas they were not changed in MAECs of wild-type and young Gla knockout mice. In addition, KCa3.1 current and the channel expression were concentration-dependently reduced in Gb3-treated MAECs. In both Gb3-treated and aged Gla knockout MAECs, extracellular signal-regulated kinase (ERK) and activator protein-1 (AP-1) were down-regulated and repressor element-1 silencing transcription factor (REST) was up-regulated. Gb3 inhibited class III phosphoinositide 3-kinase and decreased intracellular levels of phosphatidylinositol 3-phosphate [PI(3)P]. In addition, endothelium-dependent relaxation was significantly attenuated in Gb3-treated mouse aortic rings. Conclusion Gb3 accumulation reduces KCa3.1 channel expression by down-regulating ERK and AP-1 and up-regulating REST and the channel activity by decreasing intracellular levels of PI(3)P. Gb3 thereby evokes KCa3.1 channel dysfunction, and the channel dysfunction in vascular endothelial cells may contribute to vasculopathy in Fabry disease.

Aims This study examined the effects of oxidized low-density lipoprotein (LDL) and its major lipid constituent lysophosphatidylcholine (LPC) on nonselective cation (NSC) current and its inhibitory contribution to LPC-induced cytotoxicity in cultured human umbilical endothelial cells (HUVECs). Main methods Patch-clamp technique and the resazurin-based cell viability assay were used. Key findings In voltage-clamped cells, oxidized LDL or LPC slowly activated NSC current. NSC current was also activated by loading cells with Ca 2+ solution buffered at various concentrations using a patch pipette or by applying the sarcoplasmic reticulum Ca 2+ pump blocker 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ), the metabolic inhibitor CN − or the hydroperoxide donor tert -butyl hydroperoxide (TBHP). On the contrary, when intracellular Ca 2+ was strongly buffered with 12 mM BAPTA or cells were loaded with superoxide dismutase using a patch pipette, LPC or BHQ did not activate NSC current. Furthermore, NSC current activated by LPC, TBHP or CN − was inhibited by the antioxidant tempol or extracellular Ca 2+ depletion and NSC current activated by intracellular Ca 2+ was further augmented by oxidized LDL or LPC. LPC or oxidized LDL released Ca 2+ from intracellular stores and further enhanced store-operated Ca 2+ entry. LPC-induced cytotoxicity was augmented by inhibiting Ca 2+ influx and NO synthesis. Significance Oxidized LDL or its main component LPC activated Ca 2+ -permeable NSC current via releasing Ca 2+ from intracellular stores and producing ROS and thereby increased Ca 2+ influx. Ca 2+ influx through NSC channel might protect endothelial cells by producing NO.