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Physiological roles of Cl−, a major anion in the body, are not well known compared with those of cations. This review article introduces: (1) roles of Cl− in bodily and cellular functions; (2) the range of cytosolic Cl− concentration ([Cl−]c); (3) whether [Cl−]c could change with cell volume change under an isosmotic condition; (4) whether [Cl−]c could change under conditions where multiple Cl− transporters and channels contribute to Cl− influx and efflux in an isosmotic state; (5) whether the change in [Cl−]c could be large enough to act as signals; (6) effects of Cl− on cytoskeletal tubulin polymerization through inhibition of GTPase activity and tubulin polymerization-dependent biological activity; (7) roles of cytosolic Cl− in cell proliferation; (8) Cl−-regulatory mechanisms of ciliary motility; (9) roles of Cl− in sweet/umami taste receptors; (10) Cl−-regulatory mechanisms of with-no-lysine kinase (WNK); (11) roles of Cl− in regulation of epithelial Na+ transport; (12) relationship between roles of Cl− and H+ in body functions. [ABSTRACT FROM AUTHOR]

To define a normal range for PSA values (ng/mL) by age and create a prediction model for prostate cancer incidence. We conducted a retrospective analysis using 263,073 observations of PSA values in Japanese men aged 18–98 years (2007–2017), including healthy men and those diagnosed with prostate cancer. Percentiles for 262,639 PSA observations in healthy men aged 18–70 years were calculated and plotted to elucidate the normal fluctuation range for PSA values by age. Univariable and multivariable logistic regression analyses were performed to develop a predictive model for prostate cancer incidence. PSA levels and PSA velocity increased with age in healthy men. However, there was no difference in PSA velocity with age in men diagnosed with prostate cancer. Logistic regression analysis showed an increased risk of prostate cancer for PSA slopes ranging from 0.5 to 3.5 ng/mL/year. This study provides age-specific normal fluctuation ranges for PSA levels in men aged 18–75 years and presents a novel and personalized prediction model for prostate cancer incidence. We found that PSA slope values of > 3.5 ng/mL/year may indicate a rapid increase in PSA levels caused by pathological condition such as inflammation but are unlikely to indicate cancer risk. [ABSTRACT FROM AUTHOR]

Transient receptor potential vanilloid 3 (TRPV3) belongs to the TRP ion channel super family and functions as a nonselective cation channel that is highly permeable to calcium. This channel is strongly expressed in skin keratinocytes and is involved in warmth sensation, itch, wound healing and secretion of several cytokines. Previous studies showed that anoctamin1 (ANO1), a calcium-activated chloride channel, was activated by calcium influx through TRPV1, TRPV4 or TRPA1 and that these channel interactions were important for TRP channel-mediated physiological functions. We found that ANO1 was expressed by normal human epidermal keratinocytes (NHEKs). We observed that ANO1 mediated currents upon TRPV3 activation of NHEKs and mouse skin keratinocytes. Using an in vitro wound-healing assay, we observed that either a TRPV3 blocker, an ANO1 blocker or low chloride medium inhibited cell migration and proliferation through p38 phosphorylation, leading to cell cycle arrest. These results indicated that chloride influx through ANO1 activity enhanced wound healing by keratinocytes. The chloride channel anoctamin1, upon activation by calcium influx through transient receptor potential vanilloid 3, mediates chloride influx and enhances wound healing in keratinocytes. [ABSTRACT FROM AUTHOR]

Cardiac disorders are common conditions associated with a high mortality rate. Due to their potential for causing serious symptoms, it is desirable to constantly monitor cardiac status using an accessible device such as a smartwatch. While electrocardiograms (ECGs) can make the detailed diagnosis of cardiac disorders, the examination is typically performed only once a year for each individual during health checkups, and it requires expert medical practitioners to make comprehensive judgments. Here we describe a newly developed automated system for alerting individuals about cardiac disorders solely by measuring a series of heart periods. For this purpose, we examined two metrics of heart rate variability (HRV) and analyzed 1-day ECG recordings of more than 1,000 subjects in total. We found that a metric of local variation was more efficient than conventional HRV metrics for alerting cardiac disorders, and furthermore, that a newly introduced metric of local-global variation resulted in superior capacity for discriminating between premature contraction and atrial fibrillation. Even with a 1-minute recording of heart periods, our new detection system had a diagnostic performance even better than that of the conventional analysis method applied to a 1-day recording. [ABSTRACT FROM AUTHOR]

Acetylcholine (ACh), which activates muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs), enhances airway ciliary beating by increasing the intracellular Ca2+ concentration ([Ca2+]i). The mechanisms enhancing airway ciliary beating by nAChRs have remained largely unknown, although those by mAChRs are well understood. In this study, we focused on the effects of α7-nAChRs and voltage-gated Ca2+ channels (CaVs) on the airway ciliary beating. The activities of ciliary beating were assessed by frequency (CBF, ciliary beat frequency) and amplitude (CBD, ciliary bend distance) measured by high-speed video microscopy. ACh enhanced CBF and CBD by 25% mediated by an [Ca2+]i increase stimulated by mAChRs and α7-nAChRs (a subunit of nAChR) in airway ciliary cells of mice. Experiments using PNU282987 (an agonist of α7-nAChR) and MLA (an inhibitor of α7-nAChR) revealed that CBF and CBD enhanced by α7-nAChR are approximately 50% of those enhanced by ACh. CBF, CBD, and [Ca2+]i enhanced by α7-nAChRs were inhibited by nifedipine, suggesting activation of CaVs by α7-nAChRs. Experiments using a high K+ solution with/without nifedipine (155.5 mM K+) showed that the activation of CaVs enhances CBF and CBD via an [Ca2+]i increase. Immunofluorescence and immunoblotting studies demonstrated that Cav1.2 and α7-nAChR are expressed in airway cilia. Moreover, IL-13 stimulated MLA-sensitive increases in CBF and CBD in airway ciliary cells, suggesting an autocrine regulation of ciliary beating by CaV1.2/α7-nAChR/ACh. In conclusion, a novel Ca2+ signalling pathway in airway cilia, CaV1.2/α7-nAChR, enhances CBF and CBD and activates mucociliary clearance maintaining healthy airways. [ABSTRACT FROM AUTHOR]

Background: Few studies have reported the function and activation mechanism of ANO9 in esophageal squamous cell carcinoma (ESCC). The current study aimed to investigate the role of ANO9 in the regulation of tumor progression. Methods: Knockdown experiments with human ESCC cell lines were performed using ANO9 siRNA, and the effects on cell proliferation, the cell cycle, apoptosis, and cellular movement were analyzed. Immunohistochemistry (IHC) analysis was performed on 57 primary tumor samples obtained from ESCC patients. Results: In an in vitro study, depletion of ANO9 reduced cell proliferation, invasion, and migration in KYSE150 and KYSE 790 cells. In the cell cycle analysis, depletion of ANO9 increased the number of cells in G0/G1 arrest. In addition, the knockdown of ANO9 increased apoptosis. The results of the microarray analysis indicated that various centrosome-related genes such as CEP120, CNTRL, and SPAST were up- or downregulated in ANO9-depleted KYSE150 cells. The IHC results showed that high expression of ANO9 was associated with poor prognosis. Conclusions: The results of the current study suggest that ANO9 regulates the cell cycle via centrosome-related genes in ESCC. [ABSTRACT FROM AUTHOR]

The ciliary transport is controlled by two parameters of the ciliary beating, frequency (CBF) and amplitude. In this study, we developed a novel method to measure both CBF and ciliary bend distance (CBD, an index of ciliary beating amplitude) in ciliated human nasal epithelial cells (cHNECs) in primary culture, which are prepared from patients contracting allergic rhinitis and chronic sinusitis. An application of Cl−-free NO3− solution or bumetanide (an inhibitor of Na+/K+/2Cl− cotransport), which decreases intracellular Cl− concentration ([Cl−]i), increased CBD, not CBF, at 37 °C; however, it increased both CBD and CBF at 25 °C. Conversely, addition of Cl− channel blockers (5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and 4-[[4-Oxo-2-thioxo-3-[3-trifluoromethyl]phenyl]-5-thiazolidinylidene]methyl] benzoic acid (CFTR(inh)-172)), which increase [Cl−]i, decreased both CBD and CBF, suggesting that CFTR plays a crucial role for maintaining [Cl−]i in these cells. We speculate that Cl− modulates activities of the molecular motors regulating both CBD and CBF in cHNECs. Moreover, application of the CO2/HCO3−-free solution did not change intracellular pH (pHi), and addition of an inhibitor of carbonic anhydrase (acetazolamide) sustained pHi increase induced by the NH4+ pulse, which transiently increased pHi in the absence of acetazolamide. These results indicate that the cHNEC produces a large amount of CO2, which maintains a constant pHi even under the CO2/HCO3−-free condition. [ABSTRACT FROM AUTHOR]

While cancer is commonly described as "a disease of the genes," it is also associated with massive metabolic reprogramming that is now accepted as a disease "Hallmark." This programming is complex and often involves metabolic cooperativity between cancer cells and their surrounding stroma. Indeed, there is emerging clinical evidence that interrupting a cancer's metabolic program can improve patients' outcomes. The most commonly observed and well-studied metabolic adaptation in cancers is the fermentation of glucose to lactic acid, even in the presence of oxygen, also known as "aerobic glycolysis" or the "Warburg Effect." Much has been written about the mechanisms of the Warburg effect, and this remains a topic of great debate. However, herein, we will focus on an important sequela of this metabolic program: the acidification of the tumor microenvironment. Rather than being an epiphenomenon, it is now appreciated that this acidosis is a key player in cancer somatic evolution and progression to malignancy. Adaptation to acidosis induces and selects for malignant behaviors, such as increased invasion and metastasis, chemoresistance, and inhibition of immune surveillance. However, the metabolic reprogramming that occurs during adaptation to acidosis also introduces therapeutic vulnerabilities. Thus, tumor acidosis is a relevant therapeutic target, and we describe herein four approaches to accomplish this: (1) neutralizing acid directly with buffers, (2) targeting metabolic vulnerabilities revealed by acidosis, (3) developing acid-activatable drugs and nanomedicines, and (4) inhibiting metabolic processes responsible for generating acids in the first place. [ABSTRACT FROM AUTHOR]

Background: Recent studies have described important roles for the sodium iodide symporter (NIS) in tumor behavior. The objectives of the present study were to investigate the role of NIS in the regulation of genes involved in tumor progression and the clinicopathological significance of its expression in gastric cancer (GC). Methods: In human GC cell lines, knockdown experiments were conducted using NIS siRNA, and the effects on proliferation, survival, and cellular movement were analyzed. The gene expression profiles of cells were examined using a microarray analysis. An immunohistochemical analysis was performed on 145 primary tumor samples obtained from GC patients. Results: NIS was strongly expressed in MKN45 and MKN74 cells. The depletion of NIS inhibited cell proliferation, migration, and invasion and induced apoptosis. The results of the microarray analysis revealed that various interferon (IFN) signaling-related genes, such as STAT1, STAT2, IRF1, and IFIT1, were up-regulated in NIS-depleted MKN45 cells. Furthermore, the down-regulation of NIS affected the phosphorylation of MAPKs and NF-kB. Immunohistochemical staining showed that NIS was primarily located in the cytoplasm or cell membranes of carcinoma cells, and its expression was related to the histological type or venous invasion. Prognostic analyses revealed that the strong expression of NIS was associated with shorter postoperative survival. Conclusions: These results suggest that NIS regulates tumor progression by affecting IFN signaling, and that its strong expression is related to a worse prognosis in patients with GC. These results provide an insight into the role of NIS as a mediator and/or a biomarker for GC. [ABSTRACT FROM AUTHOR]

Carbocisteine (CCis), a mucoactive agent, is widely used to improve respiratory diseases. This study demonstrated that CCis increases ciliary bend angle (CBA) by 30% and ciliary beat frequency (CBF) by 10% in mouse airway ciliary cells. These increases were induced by an elevation in intracellular pH (pHi; the pHi pathway) and a decrease in the intracellular Cl− concentration ([Cl−]i; the Cl− pathway) stimulated by CCis. The Cl− pathway, which is independent of CO2/HCO3−, increased CBA by 20%. This pathway activated Cl− release via activation of Cl− channels, leading to a decrease in [Cl−]i, and was inhibited by Cl− channel blockers (5-nitro-2-(3-phenylpropylamino) benzoic acid and CFTR(inh)-172). Under the CO2/HCO3−-free condition, the CBA increase stimulated by CCis was mimicked by the Cl−-free NO3− solution. The pHi pathway, which depends on CO2/HCO3−, increased CBF and CBA by 10%. This pathway activated HCO3− entry via Na+/HCO3− cotransport (NBC), leading to a pHi elevation, and was inhibited by 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid. The effects of CCis were not affected by a protein kinase A inhibitor (1 μM PKI-A) or Ca2+-free solution. Thus, CCis decreased [Cl−]i via activation of Cl− channels including CFTR, increasing CBA by 20%, and elevated pHi via NBC activation, increasing CBF and CBA by 10%. [ABSTRACT FROM AUTHOR]

MQAE is a ‘non-ratiometric’ chloride ion (Cl−)-quenched fluorescent indicator that is used to determine intracellular Cl− concentration ([Cl−]i). MQAE-based two-photon microscopy is reported to be a useful method to measure [Cl−]i, but it is still controversial because a change in cell volume may alter the MQAE concentration, leading to a change in the fluorescence intensity without any change in [Cl−]i. In an attempt to elucidate the effect or lack of effect of cell volume on MQAE concentration, we studied the effects of changes in cell volume, achieved by applying different levels of osmotic stress, on the intensity of MQAE fluorescence in airway ciliary cells. To study solely the effect of changes in cell volume on MQAE fluorescence intensity, i.e., excluding the effect of any change in [Cl−]i, we first conducted the experiments in a Cl−-free nitrate (NO3−) solution to substitute NO3− (non-quenching anion for MQAE fluorescence) for Cl− in the intracellular fluid. Hypo- (− 30 mM NaNO3) or hyper-osmotic stress (+ 30 mM NaNO3) effected changes in cell volume, but the stress did not result in any significant change in MQAE fluorescence intensity. The experiments were also carried out in Cl−-containing solution. Hypo-osmotic stress (− 30 mM NaCl) increased both MQAE fluorescence intensity and cell volume, while hyper-osmotic stress (+ 30 mM NaCl) decreased both of these properties. These results suggest that the osmotic stress-induced change in MQAE fluorescence intensity was caused by the change in [Cl−]i and not by the MQAE concentration. Moreover, the intracellular distribution of MQAEs was heterogeneous and not affected by the changes in osmotic stress-induced cell volume, suggesting that MQAEs are bound to un-identified subcellular structures. These bound MQAEs appear to have enabled the measurement of [Cl−]i in airway ciliary cells, even under conditions of cell volume change. [ABSTRACT FROM AUTHOR]

Background: Recent evidence suggests that the targeting of membrane proteins specifically activated in cancer stem cells (CSCs) is an important strategy for cancer therapy. The objectives of the present study were to investigate the expression and activity of ion-transport-related molecules in the CSCs of esophageal squamous cell carcinoma.Methods: Cells exhibiting strong aldehyde dehydrogenase 1 family member A1 (ALDH1A1) activity were isolated from TE8 cells by fluorescence-activated cell sorting, and CSCs were then generated with the sphere formation assay. The gene expression profiles of CSCs were examined by microarray analysis.Results: Among TE8 cells, ALDH1A1 messenger RNA and protein levels were higher in CSCs than in non-CSCs. The CSCs obtained were resistant to cisplatin and had the ability to redifferentiate. The results of the microarray analysis revealed that the expression of 50 genes encoding plasma membrane proteins was altered in CSCs, whereas that of several genes related to ion channels, including transient receptor potential vanilloid 2 (TRPV2), was upregulated. The TRPV2 inhibitor tranilast was more cytotoxic at a lower concentration in CSCs than in non-CSCs, and effectively decreased the number of tumorspheres. Furthermore, tranilast significantly decreased the cell population that strongly expressed ALDH1A1 among TE8 cells.Conclusions: The results of the present study suggest that TRPV2 is involved in the maintenance of CSCs, and that its specific inhibitor, tranilast, has potential as a targeted therapeutic agent against esophageal squamous cell carcinoma. [ABSTRACT FROM AUTHOR]

This study demonstrated that PDE1 (phosphodiesterase 1) existing in the ciliary beat frequency (CBF)-regulating metabolon regulates CBF in procaterol-stimulated lung airway ciliary cells of mouse. Procaterol (an β-agonist) increased the ciliary bend angle (CBA) and CBF via cAMP accumulation in the ciliary cells of mice: interestingly, the time course of CBF increase was slower than that of CBA increase. However, IBMX (3-isobutyl-1-methylxanthine, an inhibitor of PDE) increased CBA and CBF in an identical time course. Lowering an intracellular Ca concentration ([Ca]) caused by switching to an EGTA-containing Ca-free solution from normal one elevated the procaterol-induced increasing rate of CBF. These observations suggest that Ca-dependent PDE1 controls cAMP-stimulated CBF increase. Either application of 8MmIBMX (8-methoxymethyl-IBMX, a selective PDE1 inhibitor), BAPTA-AM (an intracellular Ca chelator), or calmidazolium (an inhibitior of calmodulin) alone increased CBA and CBF in the lung airway ciliary cells and increased cAMP contents in the isolated lung cells, and like IBMX, each application of the compound made the time courses of CBA and CBF increase stimulated by procaterol identical. The immunoelectron microscopic examinations revealed that PDE1A exists in the space between the nine doublet tubules ring and plasma membrane in the lung airway cilium, where the outer dynein arm (a molecular motor regulating CBF) functions. In conclusion, PDE1A is a key factor slowing the time course of the procaterol-induced increase in CBF via degradation of cAMP in the CBF-regulating metabolon of the mouse lung airway cilia. [ABSTRACT FROM AUTHOR]

Disseminated metastasis is associated with a poor prognosis, and its management in the peritoneal or pleural cavity is crucial in the treatment of cancer. Recent studies show that ion and water transporters play important roles in fundamental cellular functions, including the regulation of cell volume that would be involved in the cancer process. Here, we review the evidence for hypotonic treatments of cancer and evaluate the potential of the cellular physiological approach in clinical management. The regulation of extracellular osmolality is a promising method, with several studies demonstrating the cytocidal effects of hypotonic solution on cancer cells. Peritoneal lavage with distilled water (DW) during surgery is reported to improve the survival rate of patients with spontaneously ruptured hepatocellular carcinoma. The in vitro studies included in this review also indicate the cytocidal effects of hypotonic shock on esophageal, gastric, colonic, pancreatic, and liver cancer cells with several unique methods and apparatuses, such as a differential interference contrast microscope connected to a digital video camera, a high-resolution flow cytometer and re-incubation analysis. The in vivo studies demonstrate the safeness of a peritoneal injection of DW into mice and indicate that the development of dissemination nodules can be prevented by the pre-incubation of cancer cells with DW or the peritoneal injection of DW. We also demonstrate that the blockade of Cl channels/transporters enhances the cytocidal effects of hypotonic shock by inhibiting regulatory volume decrease in various cancer cells. A deeper understanding of molecular mechanisms may lead to the discovery of these cellular physiological approaches as a novel therapeutic strategy for disseminated metastasis. [ABSTRACT FROM AUTHOR]

The Raman spectroscopic method has quantitatively been applied to the analysis of local crystallographic orientation in both single-crystal hydroxyapatite and human teeth. Raman selection rules for all the vibrational modes of the hexagonal structure were expanded into explicit functions of Euler angles in space and six Raman tensor elements (RTE). A theoretical treatment has also been put forward according to the orientation distribution function (ODF) formalism, which allows one to resolve the statistical orientation patterns of the nm-sized hydroxyapatite crystallite comprised in the Raman microprobe. Close-form solutions could be obtained for the Euler angles and their statistical distributions resolved with respect to the direction of the average texture axis. Polarized Raman spectra from single-crystalline hydroxyapatite and textured polycrystalline (teeth enamel) samples were compared, and a validation of the proposed Raman method could be obtained through confirming the agreement between RTE values obtained from different samples. [ABSTRACT FROM AUTHOR]

Background: Peritoneal lavage with distilled water has been performed during colorectal cancer surgery. This study investigated the cytocidal effects of hypotonic shock in vitro and in vivo in colorectal cancer cells. Methods: Three human colorectal cancer cell lines, DLD1, HT29, and CACO2, were exposed to distilled water, and morphological changes were observed under a differential interference contrast microscope connected to a high-speed digital video camera. Cell volume changes were assessed using a high-resolution flow cytometer. Re-incubation experiments were performed to investigate the cytocidal effects of distilled water. In the in vivo experiment, cancer cells after hypotonic shock were injected intraperitoneally into mice and the degree of established peritoneal metastasis was subsequently evaluated. The effects of the blockade of Cl channels on these cells during hypotonic shock were also analyzed. Results: Morphological observations revealed a rapid cell swelling followed by cell rupture. Measurements of cell volume changes showed that mild hypotonic shock induced regulatory volume decrease (RVD) while severe hypotonic shock broke cells into fragments. Re-incubation experiments demonstrated the cytocidal effects of hypotonicity. In vivo experiments revealed the absence of peritoneal dissemination in mice in the distilled water group, and its presence in all mice in the control group. The blockade of Cl channels increased cell volume by inhibiting RVD and enhanced cytocidal effects during mild hypotonic shock. Conclusions: These results clearly support the efficacy of peritoneal lavage with distilled water during colorectal cancer surgery and suggest that regulating of Cl transport may enhance the cytocidal effects of hypotonic shock. [ABSTRACT FROM AUTHOR]

It is is well known that insulin stimulates glucose transport and epithelial Na channel (ENaC)-mediated Na reabsorption; however, the action of insulin on Cl secretion is not fully understood. In this study, we investigated the action of insulin on Na-K-2Cl cotransporter (NKCC)-mediated Cl secretion in epithelial A6 cells. Interestingly, insulin treatment remarkably enhanced the forskolin-stimulated Cl secretion associated with an increase in apical Cl conductance by upregulating mRNA expression of both CFTR and NKCC, although insulin treatment alone had no effect on the basal Cl secretion or apical Cl conductance without forskolin application. We next elucidated a role of phosphoinositide 3-kinase (PI3K) in the insulin-induced enhancement of the Cl secretion, since insulin actually activated PI3K, resulting in activation of Akt, a downstream molecule of PI3K. LY294002 (a PI3K inhibitor) reduced the Cl secretion by suppressing mRNA expression of NKCC, whereas insulin still had a stimulatory action on mRNA expression of CFTR even in the presence of LY294002. On the other hand, we found that a MEK inhibitor (PD98059) further enhanced the insulin-stimulated CFTR mRNA expression and the Cl secretion in forskolin-stimulated A6 cells and that insulin induced slight, transient activation of ERK followed by significant inactivation of ERK. These observations suggest that: (1) insulin respectively upregulates mRNA expression of NKCC and CFTR through activation of PI3K and inactivation of ERK; (2) insulin signals on mRNA expression of NKCC and CFTR are not enough to stimulate transepithelial Cl secretion, but enhance the stimulatory action of cAMP on transepithelial Cl secretion. [ABSTRACT FROM AUTHOR]

nxiety disorders are the most popular psychiatric disease in any human societies irrespective of nation, culture, religion, economics or politics. Anxiety expression mediated by the amygdala may be suppressed by signals transmitted from the prefrontal cortex and hippocampus. KF-1 is an endoplasmic reticulum (ER)-based E3-ubiquitin (Ub) ligase with a RING-H2 finger motif at the C-terminus. The kf- 1 gene expression is up-regulated in the frontal cortex and hippocampus in rats after anti-depressant treatments. The kf- 1 null mice show no apparent abnormalities, but exhibit selectively pronounced anxiety-like behaviors or increased timidity-like responses. The kf- 1 orthologous genes had been generated after the Poriferan emergence, and are found widely in all animals except insects, arachnids and threadworms such as Drosophila, Ixodes and Caenorhabditis, respectively. This suggests that the kf- 1 gene may be relevant to some biological functions characteristic to animals. Based on these observations, the Anxiety Suppressor Model has been proposed, which assumes that KF-1 Ub ligase may suppress the amygdala-mediated anxiety by degrading some anxiety promoting protein(s), such as a neurotransmitter receptor, through the ER-associated degradation pathway in the frontal cortex and hippocampus. According to this model, the emotional sensitivity to environmental stresses may be regulated by the cellular protein level of KF-1 relative to that of the putative anxiety promoter. The kf- 1 null mice should be useful in elucidating the molecular mechanisms of the anxiety regulation and for screening novel anxiolytic compounds, which may block the putative anxiety promoter. [ABSTRACT FROM AUTHOR]

Intracellular pH (pHi) after the $$ {\text{NH}}^{ + }_{4} $$ pulse addition and its removal were measured in isolated alveolar type II cells (ATII cells) using BCECF fluorescence. In the absence of $$ {\text{HCO}}^{ - }_{3} $$ , the $$ {\text{NH}}^{ + }_{4} $$ pulse addition increased pHi (alkali jump) and its removal decreased pHi (acid jump) to the control level (no overacidification). This pHi change was induced by reaction 1 $$ {\left( {{\text{NH}}_{3} + {\text{H}}^{ + } \leftrightarrow {\text{NH}}^{ + }_{4} } \right)} $$ . However, in the presence of $$ {\text{HCO}}^{ - }_{3} $$ , the $$ {\text{NH}}^{ + }_{4} $$ pulse removal decreased pHi (acid jump) with overacidification. The extent of overacidification was decreased by acetazolamide (a carbonic anhydrase inhibitor), bumetanide (an inhibitor of Na+/K+/2Cl− cotransporter [NKCC]), and NPPB (an inhibitor of Cl− channel). The $$ {\text{NH}}^{ + }_{4} $$ pulse addition led to the accumulation of $$ {\text{NH}}^{ + }_{4} $$ in ATII cells via reaction 1 and NKCC, and the $$ {\text{NH}}^{ + }_{4} $$ pulse removal induced reaction 2 $$ {\left( {{\text{NH}}^{ + }_{4} + {\text{HCO}}^{ - }_{3} \to {\text{NH}}_{3} + {\text{H}}^{ + } + {\text{HCO}}^{ - }_{3} } \right)} $$ in addition to the reversal of reaction 1. Thus, $$ {\text{NH}}^{ + }_{4} $$ that entered via NKCC reacts with $$ {\text{HCO}}^{ - }_{3} $$ (reaction 2) to produce H+, which induces overacidification in the acid jump. After the overacidification, the pHi recovery consisted of a rapid recovery (first phase) followed by a slow recovery (second phase). The first phase was inhibited by NPPB, glybenclamide, amiloride, and an Na+-free solution, and the second phase was inhibited by DIDS, MIA, and an Na+-free solution. Both phases were accelerated by a high extracellular $$ {\text{HCO}}^{ - }_{3} $$ concentration. These observations indicate that the first phase was induced by $$ {\text{HCO}}^{ - }_{3} $$ entry via Cl− channels coupled with Na+ channels activities, and that the second phase was induced by H+ extrusion via Na+/H+ exchanger and by $$ {\text{HCO}}^{ - }_{3} $$ entry via $$ {\text{Na}}^{ + } - {\text{HCO}}^{ - }_{3} $$ cotransporter. Thus, in ATII cells, $$ {\text{HCO}}^{ - }_{3} $$ entry via Cl− channels is essential for recovering pHi after overacidification during the acid jump and for removing $$ {\text{NH}}^{ + }_{4} $$ that entered via NKCC from ATII cells, suggesting $$ {\text{HCO}}^{ - }_{3} $$ -dependent NH3 excretion from lungs. [ABSTRACT FROM AUTHOR]

Objectives: We conducted phase I and II studies of biweekly docetaxel and cisplatin with concurrent radiotherapy, followed by consolidation chemotherapy with the same drugs in patients with locally advanced, unresectable non-small-cell lung cancer (NSCLC). Our objectives were to define the maximum-tolerated dose and dose-limiting toxicity (DLT) in the phase I study, and to determine the response rate, toxicity, and survival rate at the recommended dose (RD) in the phase II study. Methods: Patients with unresectable stage IIIA and IIIB NSCLC were studied. Six to eight cycles of docetaxel and cisplatin were administered at 2-week intervals. In the phase I study, patients received four dose levels: level 1, docetaxel/cisplatin=30/40 mg/m2; level 2, 35/40; level 3, 40/40; and level 4, 45/40. Radiotherapy was delivered at a rate of 2 Gy per fraction/day up to a total dose of 60 Gy over the course of 6 weeks, during the first three cycles of chemotherapy. Results: DLT comprised neutropenia at level 4 in the phase I study ( n=15), and level 3 was considered the RD. In the phase II study ( n=46), two patients had a complete response (4.3%) and 34 had a partial response (73.9%), for an overall response rate of 78.2% [95% CI (66.3–90.2%)]. The survival rate was 69.1% at 1 year and 39.6% at 2 years, with a median survival time of 19.1 months. Leukopenia, neutropenia, anemia, and radiation esophagitis were the most common toxic reactions, with Grade ≥3 reactions occurring at rates of 77, 70, 17, and 8%, respectively. Conclusion: Biweekly docetaxel and cisplatin with concurrent RT was active and well tolerated in patients with unresectable stage III NSCLC. [ABSTRACT FROM AUTHOR]

The Na transport function of alveolar epithelium represents an important mechanism for clearance of fluid in air space at birth. I observed the activity of two types of amiloride-blockable Na-permeant cation channels in the apical membrane of fetal distal lung epithelium cultured on permeable filters for 2 days after harvesting of the cells from Wistar rats of 20 days' gestation (term = 22 days). One type was a nonselective cation (NSC) channel and had a linear current/voltage ( I/V) relationship with a single-channel conductance of 26.9 ± 0.8 pS ( n = 5). The other type was highly Na selective (i.e. Na channel) and had an inwardly rectifying I/V relationship with a single-channel conductance of 11.8 ± 0.2 pS ( n = 5) around resting membrane potential. The NSC channel was more frequently observed (1.37 ± 0.15 per patch membrane; n = 73) than the Na channel (0.15 ± 0.40 per patch membrane; n = 73). However, the open probability of the NSC channel was smaller than that of the Na channel. Both types of the channels were activated by cytosolic Ca, however the sensitivity to cytosolic Ca was much higher in the Na channel than in the NSC channel. Furthermore, both types of the channels were blocked by amiloride or benzamil. The half-maximal inhibitory concentration (IC) of amiloride or benzamil of the Na channel was 1-2 μM, while that of NSC channel was less than 1 μM. Both channels were activated by insulin. [ABSTRACT FROM AUTHOR]

To clarify the dependency of the Na/K coupling of the Na,K-pump on internal Na and external K concentrations in skeletal muscle, the ouabain-induced change in membrane potential, the ouabain-induced change in Na efflux and the membrane resistance were measured at various internal Na and external K concentrations in bullfrog sartorius muscle. Upon raising the internal Na concentration from 6 mmol/kg muscle water to 20 mmol/kg muscle water, the magnitude of the ouabain-induced change in membrane potential increased about eightfold and the magnitude of the ouabain-induced change in Na efflux increased about fivefold while the membrane resistance was not significantly changed. As the external K concentration increased from 1 to 10 mM, the magnitude of the ouabain-induced change in membrane potential decreased (1/5.5 fold), while the magnitude of the ouabain-induced change in Na efflux increased (about 1.5-fold). The membrane resistance decreased upon raising the external K concentration from 1 to 10 mM (1/2-fold). These observations imply that the values of the Na/K coupling of the Na,K-pump increases upon raising the internal Na concentration and decreases upon raising the external K concentration. [ABSTRACT FROM AUTHOR]

Insulin hyperpolarized the membrane of frog skeletal muscle by stimulating the electrogenic Na,K-pump. At external K concentrations of 1,2,5 and 10 mM, both the insulin-induced hyperpolarization and the insulin-stimulated ouabain-sensitive Na efflux (an index of Na,K-pump activity) were observed. By increasing the external K concentration, the insulin-stimulated Na efflux increased, but the magnitude of the insulin-induced hyperpolarization decreased; i.e., although the activity of the insulin-stimulated Na,K-pump increased, on the contrary, the magnitude of the hyperpolarization decreased. To clarify the causes of this phenomenon, the specific membrane resistance was measured and found to decrease upon increasing the external K concentration. One of the reasons for the decrease in magnitude of the hyperpolarization is the decrease in the specific membrane resistance. However, the decrease in magnitude of the hyperpolarization with a rise of the external K concentration, which increased the insulin-stimulated Na,K-pump activity, cannot be explained only by the decrease in the specific membrane resistance. It is suggested that the decrease in magnitude of the hyperpolarization is mainly caused by a decrease in the electrogenicity of the insulin-stimulated Na,K-pump upon an increase in the external K concentration. The conclusion of the present study is that the electrogenicity of the insulin-stimulated Na,K-pump in muscles is variable and decreases with increasing the external K concentration. [ABSTRACT FROM AUTHOR]

A Na+-sensitive uptake of 3-O-methylglucose (3-O-MG), a nonmetabolized sugar, was characterized in frog skeletal muscle. A removal of Na+ from the bathing solution reduced 3-O-MG uptake, depending on the amount of Na+ removed. At a 3-O-MG concentration of 2 mM, the Na+-sensitive component of uptake in Ringer's solution was estimated to be about 26% of the total uptake. The magnitude of Na+-sensitive component sigmoidally increased with an increase of 3-O-MG in bathing solution, whereas in Na+-free Ringer's solution the uptake was proportional to the concentration. The half saturation of the Na+-sensitive component was at a 3-O-MG concentration of about 13 mM, and the Hill coefficient was 1.4 to 1.6. Phlorizin (5 mM), a potent inhibitor specific for Na+-coupled glucose transport, reduced the uptake in a solution containing Na+ to the level in Na+-free Ringer's solution. Glucose of concentrations higher than 20 mM suppressed 3-O-MG uptake to a level slightly lower than that in Na+-free Ringer's solution. These observations indicate that there are Na+-coupled sugar transport systems in frog skeletal muscle which are shared by both glucose and 3-O-MG. [ABSTRACT FROM AUTHOR]

The first step in net active transepithelial transport of sodium in tight epithelia is mediated by the amiloride-blockable sodium channel in the apical membrane. This sodium channel is the primary site for discretionary control of total body sodium and, therefore, investigating its regulatory mechanisms is important to our understanding of the physiology of fluid and electrolyte balance. Because essentially all of the regulatory sites on the channel are on the intracellular surface, patch clamp methods have proven extremely useful in the electrophysiological characterization of the sodium channel by isolating it from other channel proteins in the epithelial membrane and by allowing access to the intracellular surface of the protein. We have examined three different regulatory mechanisms. (1) Inhibition of channel activity by activation of protein kinase C; (2) activation of the channel by agents which activate G-proteins; and (3) modulation of channel kinetics and channel number by mineralocorticoids. Activation of protein kinase C by phorbol esters or synthetic diacylglycerols reduces the open probability of sodium channels. Protein kinase C can be activated in a physiological context by enhancing apical sodium entry. Actions which reduce sodium entry (low luminal sodium concentrations or the apical application of amiloride) increase channel open probability. The link between sodium entry and activation of protein kinase C appears to be mediated by intracellular calcium activity linked to sodium via a sodium/calcium exchange system. Thus, the intracellular sodium concentration is coupled to sodium entry in a negative feedback loop which promotes constant total entry of sodium. Activation of G-proteins by pertussis toxin greatly increases the open probability of sodium channels. Since channels can also be activated by pertussis toxin or GTP gamma S in excised patches, the G-protein appears to be closely linked in the apical membrane to the sodium channel protein itself. The mechanism for activation of this apical G-protein, when most hormonal and transmitter receptors are physically located on the basolateral membrane, is unclear. Mineralocorticoids such as aldosterone have at least two distinct effects. First, as expected, increasing levels of aldosterone increase the density of functional channels detectable in the apical membrane. Second, contrary to expectations, application of aldosterone increases the open probability of sodium channels. Thus aldosterone promotes the functional appearance of new sodium channels and promotes increased sodium entry through both new and pre-existant channels. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the roles of tyrosine kinase (TK) in the insulin action on cell volume in fetal rat (20-day gestational age) type II pneumocyte. Insulin (100 nmol/l) increased cell volume, and this insulin (100 nmol/l) action was completely blocked by 50 μmol/l bumetanide (BMT) and 10 μmol/l amiloride (AML). This observation indicates that 100 nmol/l insulin activates BMT-sensitive Na/K/2Cl cotransporter and AML-sensitive pathways. The stimulatory action of 100 nmol/l insulin on BMT-sensitive Na/K/2Cl cotransporter was completely abolished by 10 μmol/l lavendustin A (LAV-A, an inhibitor of TK), however 100 nmol/l insulin could stimulate AML-sensitive pathways even in LAV-A (10 μmol/l)-treated cells. These observations indicate that the insulin (100 nmol/l) action on the BMT-sensitive Na/K/2Cl cotransporter is mediated through TK-dependent pathways, while 100 nmol/l insulin requires a TK-independent pathway to show the stimulatory action on the AML-sensitive pathways. From these observations we conclude that TK-dependent and -independent pathways are involved in the insulin (100 nmol/l) signaling in fetal rat type II pneumocyte. [ABSTRACT FROM AUTHOR]

Background: Transient receptor potential vanilloid 2 (TRPV2) was recently shown to be involved in migrant potentials. The present study aimed to investigate its role in esophageal squamous cell carcinoma (ESCC). Methods: Knockdown experiments were conducted using TRPV2 siRNA in human ESCC cell lines, and anti-tumor effects were analyzed. The gene expression profiles of cells were analyzed using a microarray method. An immunohistochemical staining was performed on 62 primary tumor samples. Results: TRPV2 overexpression was observed in TE15 and KYSE170 cells. TRPV2 depletion suppressed proliferation, cell cycle progression, and invasion/migration ability, and induced apoptosis. A pathway analysis of microarray data showed that TRPV2 depletion down-regulated WNT/β-catenin signaling-related genes and basal cell carcinoma signaling-related genes. The suppression of tumor functions, such as proliferation, invasion, and angiogenesis, was predicted in the ontology analysis. Immunohistochemical analysis revealed a correlation between strong TRPV2 expression and a poor prognosis in ESCC patients. Conclusion: The present results suggest that TRPV2 regulates cancer progression by affecting WNT/β-catenin or basal cell carcinoma signaling, and that TRPV2 strong expression is associated with a worse prognosis in ESCC patients. These results provide an insight into the role of TRPV2 as a novel therapeutic target or biomarker for ESCC. [ABSTRACT FROM AUTHOR]