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An application of CO 2 /HCO 3 - -free solution (Zero-CO 2 ) did not increase intracellular pH (pH i ) in ciliated human nasal epithelial cells (c-hNECs), leading to no increase in frequency (CBF) or amplitude (CBA) of the ciliary beating. This study demonstrated that the pH i of c-hNECs expressing carbonic anhydrase IV (CAIV) is high (7.64), while the pH i of ciliated human bronchial epithelial cells (c-hBECs) expressing no CAIV is low (7.10). An extremely high pH i of c-hNECs caused pH i , CBF and CBA to decrease upon Zero-CO 2 application, while a low pH i of c-hBECs caused them to increase. An extremely high pH i was generated by a high rate of HCO 3 - influx via interactions between CAIV and Na + /HCO 3 - cotransport (NBC) in c-hNECs. An NBC inhibitor (S0859) decreased pH i , CBF and CBA and increased CBF and CBA in c-hNECs upon Zero-CO 2 application. In conclusion, the interactions of CAIV and NBC maximize HCO 3 - influx to increase pH i in c-hNECs. This novel mechanism causes pH i to decrease, leading to no increase in CBF and CBA in c-hNECs upon Zero-CO 2 application, and appears to play a crucial role in maintaining pH i , CBF and CBA in c-hNECs periodically exposed to air (0.04% CO 2 ) with respiration.

Single cilia, 100 nm in diameter and 10 µm in length, were isolated from mouse tracheae with Triton X-100 (0.02%) treatment, and the effects of pH on ciliary beating were examined by measuring the ciliary beat frequency (CBF) and the ciliary bend distance (CBD-an index of amplitude) using a high-speed video microscope (250 fps). ATP (2.5 mM) plus 8Br-cAMP (10 µM) reactivated the CBF and CBD in the isolated cilia, similar to the cilia of in vivo tracheae. In the reactivated isolated cilia, an elevation in pH from 7.0 to 8.0 increased the CBF from 3 to 15 Hz and the CBD from 0.6 to 1.5 µm. The pH elevation also increased the velocity of the effective stroke; however, it did not increase the recovery stroke, and, moreover, it decreased the intervals between beats. This indicates that H + (pH i ) directly acts on the axonemal machinery to regulate CBF and CBD. In isolated cilia priorly treated with 1 µM PKI-amide (a PKA inhibitor), 8Br-cAMP did not increase the CBF or CBD in the ATP-stimulated isolated cilia. pH modulates the PKA signal, which enhances the axonemal beating generated by the ATP-activated inner and outer dyneins.

Ambroxol (ABX), a frequently prescribed secretolytic agent which enhances the ciliary beat frequency (CBF) and ciliary bend angle (CBA, an index of amplitude) by 30%, activates a voltage-dependent Ca 2+ channel (Ca V 1.2) and a small transient Ca 2+ release in the ciliated lung airway epithelial cells (c-LAECs) of mice. The activation of Ca V 1.2 alone enhanced the CBF and CBA by 20%, mediated by a pH i increase i and a [Cl - ] i decrease in the c-LAECs. The increase in pH i , which was induced by the activation of the Na + -HCO 3 - cotransporter (NBC), enhanced the CBF (by 30%) and CBA (by 15-20%), and a decrease in [Cl - ] i , which was induced by the Cl - release via anoctamine 1 (ANO1), enhanced the CBA (by 10-15%). While a Ca 2+ -free solution or nifedipine (an inhibitor of Ca V 1.2) inhibited 70% of the CBF and CBA enhancement using ABX, Ca V 1.2 enhanced most of the CBF and CBA increases using ABX. The activation of the Ca V 1.2 existing in the cilia stimulates the NBC to increase pH i and ANO1 to decrease the [Cl - ] i in the c-LAECs. In conclusion, the pH i increase and the [Cl - ] i decrease enhanced the CBF and CBA in the ABX-stimulated c-LAECs.

Ambroxol (ABX) facilitates the mucociliary clearance (MC) by enhancing ciliary beating in airways. In this study, we focused on airway ciliary beating enhanced by ABX. However, little is known about the ABX-stimulated Ca 2+ signalling activating airway ciliary beating. Airway ciliated cells isolated from mice lungs were observed by a high-speed video microscope, and the activities of beating cilia were assessed by CBF (ciliary beat frequency) and CBD (ciliary bend distance, an index of amplitude). ABX (10 μM) enhanced the CBF and CBD by 30%, and the enhancement was inhibited by nifedipine (20 μM, a L-type voltage-gated Ca 2+ channel (Ca V ) inhibitor), or a Ca 2+ -free solution (approximately 50%). Pre-treatment with BAPTA-AM (10 μM, a chelator of intracellular Ca 2+ ) abolished ABX-stimulated increases in CBF, CBD and [Ca 2+ ] i . Thus, ABX increases [Ca 2+ ] i (intracellular Ca 2+ concentration) by stimulating Ca 2+ release from the internal stores and nifedipine-sensitive Ca 2+ entry. A previous study demonstrated the expression of Ca V 1.2 in airway cilia. ABX enhanced CBF, CBD and [Ca 2+ ] i even in a high extracellular K + concentration (155.5 mM), suggesting that it activates Ca V 1.2 except by depolarization. These enhancements were inhibited by nifedipine. In conclusion, ABX, which increases [Ca 2+ ] i by stimulating Ca 2+ release from internal stores and Ca 2+ entry through Ca V 1.2s, enhanced CBF and CBD in airway ciliated cells. ABX is a novel agonist that modulates Ca V 1.2 of airway beating cilia to enhance CBF and CBD., Competing Interests: Declaration of competing interest This work was funded by Teijin Pharma Limited, Japan to TN and YM., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The present study tried to clarify if mumefural would prevent hyperglycemia, one of the typical symptoms of type 2 diabetes mellitus (T2DM), since mumefural is an extract from Japanese apricots preventing hyperglycemia. To clarify if mumefural would prevent T2DM pathogenesis, we used Otsuka Long-Evans Tokushima fatty (OLETF) rats, T2DM model. Mumefural diminished hyperglycemia, HOMA-IR and plasma triglyceride concentration in OLETF rats under fasting conditions. In addition, mumefural elevated protein expression of sodium-coupled monocarboxylate transporter 1 (SMCT1) in the distal colon participating in absorption of weak organic acids, which behave as bases but not acids after absorption into the body. Mumefural also increased the interstitial fluid pH around the brain hippocampus lowered in OLETF rats compared with non-T2DM LETO rats used as control for OLETF rats. Amyloid-beta accumulation in the brain decreased in accordance with the pH elevation. On the one hand, mumefural didn't affect plasma concentrations of glucagon, GLP-1, GIP or PYY under fasting conditions. Taken together, these observations indicate that: 1) mumefural would be a useful functional food improving hyperglycemia, insulin resistance and the lowered interstitial fluid pH in T2DM; 2) the interstitial fluid pH would be one of key factors influencing the accumulation of amyloid-beta.

Ependymal cilia play pivotal roles in cerebrospinal fluid flow. In the primary culture system, undifferentiated glial cells differentiate well into ependymal multiciliated cells (MCCs) in the absence of fetal bovine serum (FBS). However, the substances included in FBS which inhibit this differentiation process have not been clarified yet. Here, we constructed the polarized primary culture system of ependymal cells using a permeable filter in which they retained ciliary movement. We found that transforming growth factor-β1 (TGF-β1) as well as Bone morphogenetic protein (BMP)-2 inhibited the differentiation with ciliary movement. The inhibition on the differentiation by FBS was recovered by the TGF-β1 and BMP-2 inhibitors in combination.

Acetylcholine (ACh), which activates muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs), enhances airway ciliary beating by increasing the intracellular Ca 2+ concentration ([Ca 2+ ] 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 Ca 2+ channels (Ca V s) 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 [Ca 2+ ] 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 [Ca 2+ ] i enhanced by α7-nAChRs were inhibited by nifedipine, suggesting activation of Ca V s by α7-nAChRs. Experiments using a high K + solution with/without nifedipine (155.5 mM K + ) showed that the activation of Ca V s enhances CBF and CBD via an [Ca 2+ ] 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 Ca V 1.2/α7-nAChR/ACh. In conclusion, a novel Ca 2+ signalling pathway in airway cilia, Ca V 1.2/α7-nAChR, enhances CBF and CBD and activates mucociliary clearance maintaining healthy airways., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Mucociliary clearance, which is conducted by beating cilia cooperating with the surface mucous layer, is a major host defense mechanism of the airway epithelium. Ezrin, a crosslinker between membrane proteins and the actin cytoskeleton, is located in microvilli and around the basal bodies in airway ciliary cells. It is also likely that ezrin plays an important role in apical localization of β2 adrenergic receptor (β2AR) in airway ciliary cells. Here, we studied the physiological roles of ezrin by using trachea and airway epithelial cells prepared from ezrin-knockdown (Vil2kd/kd) mice. The trachea and airway ciliary cells of Vil2kd/kd mice presented a normal morphology and basal body orientation, suggesting that ezrin is not directly involved in development and planar cell polarity of cilia. Procaterol stimulates ciliary beating (frequency and amplitude) via β2AR in the airway ciliary cells. In the Vil2kd/kd mice, airway ciliary beating stimulated with procaterol was partly inhibited due to the impairment of cell surface expression of β2AR. These results suggest that ezrin regulates the beating of airway ciliary cells by promoting the apical surface localization of β2AR. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The HCO 3 - concentration in venous serum ([HCO 3 - ] s ) is a factor commonly used for detecting the body pH and metabolic conditions. To exactly detect [HCO 3 - ] s , the venous CO 2 pressure should be kept as it is in the vein. The [HCO 3 - ] s measurement is technically complicated to apply for huge numbers of almost heathy persons taking only basic medical examinations. The summation of [HCO 3 - ] s and the venous serum Cl - concentration ([Cl - ] s ) is approximately constant; therefore, we studied if [Cl - ] s could be a marker detecting metabolic conditions instead of [HCO 3 - ] s . Venous blood was obtained from persons taking basic medical examinations (the number of persons = 107,630). Older persons showed higher values of [Cl - ] s , fasting blood sugar (FBS), and glycated hemoglobin (HbA1c) than younger ones. [Cl - ] s showed positive correlation to age and negative correlation to FBS and HBA1c. The negative correlation of [Cl - ] s to FBS/HbA1c was obvious in persons with high FBS/HbA1c, leading us to an idea that persons with high FBS/HbA1c show high [HCO 3 - ] s , which might be caused by low activity of carbonic anhydrase in the lung observed in persons with diabetes mellitus under acidotic conditions. Taken together, an easily measured serum electrolyte, [Cl - ] s , could be a useful marker estimating metabolic conditions.

New Findings: What is the central question of this study? Arachidonic acid (AA) stimulates NO production in antral mucous cells without any increase in [Ca 2+ ] i . Given that the intracellular AA concentration is too low to measure, the relationship between AA accumulation and NO production remains uncertain. Is AA accumulation a key step for NO production? What is the main finding and its importance? We demonstrated that AA accumulation is a key step for NO production. The amount of AA released could be measured using fluorescence-HPLC. The intracellular AA concentration was maintained at < 1 μM. Nitric oxide is produced by AA accumulation in antral mucous cells, not as a direct effect of [Ca 2+ ] i ., Abstract: In the present study, we demonstrate that NO production is stimulated by an accumulation of arachidonic acid (AA) mediated via peroxisome proliferation-activated receptor α (PPARα) and that the NO produced enhances Ca 2+ -regulated exocytosis in ACh-stimulated antral mucous cells. The amount of AA released from the antral mucosa, measured by fluorescence high-performance liquid chromatography (F-HPLC), was increased by addition of ionomycin (10 μM) or ACh, suggesting that AA accumulation is stimulated by an increase in [Ca 2+ ] i . The AA production was inhibited by an inhibitor of cytosolic phospholipase A2 (cPLA2-inhα). GW6471 (a PPARα inhibitor) and cPLA2-inhα inhibited NO synthesis stimulated by ACh. Moreover, indomethacin, an inhibitor of cyclooxygenase, stimulated AA accumulation and NO production. However, acetylsalicylic acid did not stimulate AA production and NO synthesis. An analogue of AA (AACOCF3) alone stimulated NO synthesis, which was inhibited by GW6471. In antral mucous cells, indomethacin enhanced Ca 2+ -regulated exocytosis by increasing NO via PPARα, and the enhancement was abolished by GW6471 and cPLA2-inhα. Thus, AA produced via PLA2 activation is the key step for NO synthesis in ACh-stimulated antral mucous cells and plays important roles in maintaining antral mucous secretion, especially in Ca 2+ -regulated exocytosis., (© 2021 The Authors. Experimental Physiology © 2021 The Physiological Society.)

Moesin is a member of the ezrin, radixin and moesin (ERM) proteins that are involved in the formation and/or maintenance of cortical actin organization through their cross-linking activity between actin filaments and proteins located on the plasma membranes as well as through regulation of small GTPase activities. Microglia, immune cells in the central nervous system, show dynamic reorganization of the actin cytoskeleton in their process elongation and retraction as well as phagocytosis and migration. In microglia, moesin is the predominant ERM protein. Here, we show that microglial activation after systemic lipopolysaccharide application is partly inhibited in moesin knockout (Msn-KO) mice. We prepared primary microglia from wild-type and Msn-KO mice, and studied them to compare their phenotypes accompanying morphological changes and reorganization of the actin cytoskeleton induced by UDP-stimulated phagocytosis and ADP-stimulated migration. The Msn-KO microglia showed higher phagocytotic activity in the absence of UDP, which was not further increased by the treatment with UDP. They also exhibited decreased ADP-stimulated migration activities compared with the wild-type microglia. However, the Msn-KO microglia retained their ability to secrete tumor necrosis factor α and nitric oxide in response to lipopolysaccharide.

Small inhaled particles, which are entrapped by the mucous layer that is maintained by mucous secretion via mucin exocytosis and fluid secretion, are removed from the nasal cavity by beating cilia. The functional activities of beating cilia are assessed by their frequency and the amplitude. Nasal ciliary beating is controlled by intracellular ions (Ca 2+ , H + and Cl - ), and is enhanced by a decreased concentration of intracellular Cl - ([Cl - ] i ) in ciliated human nasal epithelial cells (cHNECs) in primary culture, which increases the ciliary beat amplitude. A novel method to measure both ciliary beat frequency (CBF) and ciliary beat distance (CBD, an index of ciliary beat amplitude) in cHNECs has been developed using high-speed video microscopy, which revealed that a decrease in [Cl - ] i increased CBD, but not CBF, and an increase in [Cl - ] i decreased both CBD and CBF. Thus, [Cl - ] i inhibits ciliary beating in cHNECs, suggesting that axonemal structures controlling CBD and CBF may have Cl - sensors and be regulated by [Cl - ] i . These observations indicate that the activation of Cl - secretion stimulates ciliary beating (increased CBD) mediated via a decrease in [Cl - ] i in cHNECs. Thus, [Cl - ] i is critical for controlling ciliary beating in cHNECs. This review introduces the concept of Cl - regulation of ciliary beating in cHNECs.

Objective: Carbocisteine (CCis), a mucoactive agent, is used to improve the symptoms of sinonasal diseases. However, the effect of CCis on nasal ciliary beating remains uncertain. We examined the effects of CCis on ciliary beat distance (CBD, an index of amplitude), and ciliary beat frequency (CBF) in ciliated human nasal epithelial cells (cHNECs) in primary culture., Methods: The cHNECs were prepared from the nasal tissue resected from patients required surgery for chronic sinusitis (CS) or allergic rhinitis (AR). CBD and CBF were measured using videomicroscopy equipped with a high-speed camera., Results: CCis increased CBD by 30%, but not CBF, and decreased intracellular Cl - concentration ([Cl - ] i ) in cHNECs. The CCis' actions were mimicked by the Cl - -free NO 3 - solution. In contrast, prior treatment of NPPB (20 μM) or CFTR(inh)-172 (1 μM), which increased [Cl - ] i by 20%, decreased CBF by 10% and CBD by 25% and inhibited the CCis' actions. However, prior treatment of T16Ainh-A01 (10 μM) did not inhibit the CCis' actions, although it decreased [Cl - ] i by 10% and CBD by 15%. Thus, CCis stimulates Cl - channels including cystic fibrosis transmembrane conductance regulator (CFTR). Moreover, CCis enhanced the transport of microbeads driven by the beating cilia in cHNECs. The CCis actions were similar in cHNECs from both types of pateints., Conclusion: CCis increased CBD by 30% in cHNECs via an [Cl - ] i decrease stimulated by activation of Cl - channels, including CFTR. CCis may stimulate nasal mucociliary clearance by increasing CBD in patients contracting CS or AR., Level of Evidence: NA. Laryngoscope, 130:E289-E297, 2020., (© 2019 The American Laryngological, Rhinological and Otological Society, Inc.)

In Ts1Rhr, a Down syndrome model mouse, the airway ciliary beatings are impaired; that is, decreases in ciliary beat frequency (CBF) and ciliary bend angle (CBA, an index of ciliary beat amplitude)). A resumption to two copies of the Pcp4 gene on the Ts1Rhr trisomic segment (Ts1Rhr: Pcp4 +/+/- ) rescues the decreases in CBF and CBA that occur in Ts1Rhr. In airway cilia, upon stimulation with procaterol (a β 2 -agonist), the CBF increase is slower over the time course than the CBA increase because of cAMP degradation by Ca 2+ /calmodulin-dependent phosphodiesterase 1 (PDE1) existing in the metabolon regulating CBF. In Ts1Rhr, procaterol-stimulated CBF increase was much slower over the time course than in the wild-type mouse (Wt) or Ts1Rhr: Pcp4 +/+/- . However, in the presence of 8MmIBMX (8-methoxymethyl isobutylmethyl xanthine, an inhibitor of PDE1) or calmidazolium (an inhibitor of calmodulin), in both Wt and Ts1Rhr, procaterol stimulates CBF and CBA increases over a similar time course. Measurements of cAMP revealed that the cAMP contents were lower in Ts1Rhr than in Wt or in Ts1Rhr: Pcp4 +/+/- , suggesting the activation of PDE1A that is present in Ts1Rhr airway cilia. Measurements of the intracellular Ca 2+ concentration ([Ca 2+ ] i ) in airway ciliary cells revealed that temperature (increasing from 25 to 37 °C) or 4αPDD (a selective transient receptor potential vanilloid 4 (TRPV4) agonist) stimulates a larger [Ca 2+ ] i increase in Ts1Rhr than in Wt or Ts1Rhr: Pcp4 +/+/- . In airway ciliary cells of Ts1Rhr, Pcp4 -dose dependent activation of TRPV4 appears to induce an increase in the basal [Ca 2+ ] i . In early embryonic day mice, a basal [Ca 2+ ] i increased by PCP4 expressed may affect axonemal regulatory complexes regulated by the Ca 2+ -signal in Ts1Rhr, leading to a decrease in the basal CBF and CBA of airway cilia.

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 NO 3 - 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 CO 2 /HCO 3 - -free solution did not change intracellular pH (pH i ), and addition of an inhibitor of carbonic anhydrase (acetazolamide) sustained pH i increase induced by the NH 4 + pulse, which transiently increased pH i in the absence of acetazolamide. These results indicate that the cHNEC produces a large amount of CO 2 , which maintains a constant pH i even under the CO 2 /HCO 3 - -free condition.

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 (pH i ; the pH i pathway) and a decrease in the intracellular Cl - concentration ([Cl - ] i ; the Cl - pathway) stimulated by CCis. The Cl - pathway, which is independent of CO 2 /HCO 3 - , 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 CO 2 /HCO 3 - -free condition, the CBA increase stimulated by CCis was mimicked by the Cl - -free NO 3 - solution. The pH i pathway, which depends on CO 2 /HCO 3 - , increased CBF and CBA by 10%. This pathway activated HCO 3 - entry via Na + /HCO 3 - cotransport (NBC), leading to a pH i 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 Ca 2+ -free solution. Thus, CCis decreased [Cl - ] i via activation of Cl - channels including CFTR, increasing CBA by 20%, and elevated pH i via NBC activation, increasing CBF and CBA by 10%.

Ciliary beating frequency (CBF) was investigated in ciliated nasal epithelial cells (cMNECs) isolated from mice using video microscopy equipped with a high-speed camera. In cMNECs, a spontaneous CBF oscillation was observed. The CBF oscillation was abolished by BAPTA-AM but not by Ca 2+ -free solution. The addition of thapsigargin, which depletes Ca 2+ from internal stores, also abolished CBF oscillation. Moreover, the intracellular Ca 2+ concentration [Ca 2+ ] i , spontaneously oscillated even with the Ca 2+ -free solution. Moreover, 2APB (an inhibitor of the IP 3 receptor) abolished CBF oscillation in cMNECs. Overall, these findings suggest that the CBF oscillation in cMNECs is triggered by the release of Ca 2+ from the IP 3 -sensitive internal stores. Moreover, IBMX, an inhibitor of phosphodiesterase, did not affect CBF oscillation in cMNECs, although it slightly increased CBF. These results suggest that CBF oscillations were induced by [Ca 2+ ] i oscillation controlled via the release of Ca 2+ from IP 3 -sensitive stores, rather than via cAMP accumulation. CBF oscillation possibly plays a crucial role in maintaining an efficient mucociliary clearance in the nasal epithelia., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The effects of the isoflavone daidzein on the ciliary beat distance (CBD, which is a parameter assessing the amplitude of ciliary beating) and the ciliary beat frequency (CBF) were examined in ciliated human nasal epithelial cells (cHNECs) in primary culture. Daidzein decreased [Cl - ] i and enhanced CBD in cHNECs. The CBD increase that was stimulated by daidzein was mimicked by Cl - -free NO₃ - solution and bumetanide (an inhibitor of Na⁺/K⁺/2Cl - cotransport), both of which decreased [Cl - ] i. Moreover, the CBD increase was inhibited by 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, a Cl - channel blocker), which increased [Cl - ] i . CBF was also decreased by NPPB. The rate of [Cl - ] i decrease evoked by Cl - -free NO₃ - solution was enhanced by daidzein. These results suggest that daidzein activates Cl - channels in cHNECs. Moreover, daidzein enhanced the microbead transport driven by beating cilia in the cell sheet of cHNECs, suggesting that an increase in CBD enhances ciliary transport. An [Cl - ] i decrease enhanced CBD, but not CBF, in cHNECs at 37 °C, although it enhanced both at 25 °C. Intracellular Cl - affects both CBD and CBF in a temperature-dependent manner. In conclusion, daidzein, which activates Cl - channels to decrease [Cl - ] i , stimulated CBD increase in cHNECs at 37 °C. CBD is a crucial factor that can increase ciliary transport in the airways under physiological conditions.

New Findings: What is the central question of this study? The ciliary beat frequency (CBF) of the airway is controlled by [Ca 2+ ] i . However, the effects of a reduction in [Ca 2+ ] i on CBF are still controversial (an increase, a decrease or no change). What is the main finding and its importance? This study demonstrated that [Ca 2+ ] i directly regulates CBF (direct action) and also indirectly regulates CBF via cAMP accumulation controlled by Ca 2+ -dependent PDE1 activity (indirect action). The final CBF is determined by the balance of direct and indirect actions. PDE1 plays crucial roles in the regulation of airway CBF. ABSTRACT: [Ca 2+ ] i plays crucial roles in the regulation of ciliary beat frequency (CBF) and ciliary bend angle (CBA) of airway cilia. Moreover, Ca 2+ -dependent PDE1A existing in the CBF-regulating metabolon of cilia modifies the CBF by regulating the cAMP accumulation. This study demonstrated that the CBF is regulated by a direct and an indirect action of [Ca 2+ ] i ; the direct action changes CBF mediated via [Ca 2+ ] i , and the indirect action changes CBF mediated via cAMP, the accumulation of which is controlled by PDE1 activity. Upon reducing [Ca 2+ ] i to various levels, the direct action decreases CBF and the indirect action increases CBF. The final CBF is determined by the extent of cAMP accumulation, which is determined by the amount of inhibition of PDE1 activity, dependent on a reduction in [Ca 2+ ] i ; a slight decrease induced by a nominally Ca 2+ -free solution (no cAMP accumulation via PDE1) decreases CBF, and an extreme decrease induced by 50 μm BAPTA-AM increases CBF via cAMP accumulation by inhibiting PDE1 in a similar manner to a PDE1 inhibitor (8MmIBMX). The increase in CBA in response to a reduction in [Ca 2+ ] i is smaller than the increase in CBF, because no PDE1A exists in the CBA-regulating metabolon. On the contrary, an increase in [Ca 2+ ] i induced by ionomycin, which decreases cAMP accumulation by PDE1A activation, caused a slower procaterol-stimulated increase in CBF than that decreased by a Ca 2+ -free solution. A decrease in [Ca 2+ ] i stimulates cAMP accumulation, whereas an increase in [Ca 2+ ] i inhibits cAMP accumulation in airway ciliary cells. Thus, changes in [Ca 2+ ] i modulate CBF and CBA via cAMP accumulation by controlling the activity of PDE1., (© 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.)

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 (NO 3 - ) solution to substitute NO 3 - (non-quenching anion for MQAE fluorescence) for Cl - in the intracellular fluid. Hypo- (- 30 mM NaNO 3 ) or hyper-osmotic stress (+ 30 mM NaNO 3 ) 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.

Sei-hai-to (TJ-90, Qing Fei Tang), a Chinese traditional medicine, increases ciliary beat frequency (CBF) and ciliary bend angle (CBA) mediated via cAMP (3',5'-cyclic adenosine monophosphate) accumulation modulated by Ca 2+ -activated phosphodiesterase 1 (PDE1A). A high concentration of TJ-90 (≥40 μg/mL) induced two types of CBF increases, a transient increase (an initial increase, followed by a decrease) and a sustained increase without any decline, while it only sustained the CBA increase. Upon inhibiting increases in intracellular Ca 2+ concentration ([Ca 2+ ] i ) by 10 μM BAPTA-AM (Ca 2+ -chelator, 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) or Ca 2+ /calmodulin-dependent PDE1 by 8MmIBMX (a selective PDE1 inhibitor), TJ-90 (400 μg/mL) induced only the sustained CBF increase without any transient CBF increase. The two types of the CBF increase (the transient increase and the sustained increase) induced by TJ-90 (≥40 μg/mL) were mimicked by the stimulation with both procaterol (100 pM) and ionomycin (500 nM). Thus, TJ-90 stimulates small increases in the intracellular cAMP concentration ([cAMP] i ) and [Ca 2+ ] i in airway ciliary cells of mice. These small increases in [cAMP] i and [Ca 2+ ] i cause inducing a transient CBF increase or a sustained CBF increase in an airway ciliary cells, depending on the dominant signal, Ca 2+ -signal, or cAMP-signal., Competing Interests: The authors declare no conflict of interest.

Quercetin has multiple potential to control various cell function keeping our body condition healthy. In this review article, we describe the molecular mechanism on how quercetin exerts its action on blood pressure, neurite elongation and epithelial ion transport based from a viewpoint of cytosolic Cl- environments, which is recently recognized as an important signaling factor in various types of cells. Recent studies show various roles of cytosolic Cl- in regulation of blood pressure and neurite elongation, and prevention from bacterial and viral infection. We have found the stimulatory action of quercetin on Cl- transporter, Na+-K+-2Cl- cotransporter 1 (NKCC1; an isoform of NKCC), which has been recognized as one of the most interesting, fundamental actions of quercetin. In this review article, based on this stimulatory action of quercetin on NKCC1, we introduce the molecular mechanism of quercetin on: 1) blood pressure, 2) neurite elongation, and 3) epithelial Cl- secretion including tight junction forming in epithelial tissues. 1) Quercetin induces elevation of the cytosolic Cl- concentration via activation of NKCC1, leading to anti-hypertensive action by diminishing expression of epithelial Na+ channel (ENaC), a key ion channel involved in renal Na+ reabsorption, while quercetin has no effects on the blood pressure with normal salt intake. 2) Quercetin also has stimulatory effects on neurite elongation by elevating the cytosolic Cl- concentration via activation of NKCC1 due to tubulin polymerization facilitated through Cl--induced inhibition of GTPase. 3) Further, in lung airway epithelia quercetin stimulates Cl- secretion by increasing the driving force for Cl- secretion via elevation of the cytosolic Cl- concentration: this leads to water secretion, participating in prevention of our body from bacterial and viral infection. In addition to transcellular ion transport, quercetin regulates tight junction function via enhancement of tight junction integrity by modulating expression and assembling tight junction-forming proteins. Based on these observations, it is concluded that quercetin is a useful medicinal compound keeping our body to be in healthy condition., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The detailed single-channel gating kinetics of mouse pannexin 1 (mPanx1) remains unknown, although mPanx1 is reported to be a voltage-activated anion-selective channel. We investigated characteristics of single-channel conductances and opening and closing rates of mPanx1 using patch-clamp techniques. The unitary current of mPanx1 shows outward rectification with single-channel conductances of ~20 pS for inward currents and ~80 pS for outward currents. The channel open time for outward currents (Cl - influx) increases linearly as the amplitude of single channel currents increases, while the open time for inward currents (Cl - efflux) is constant irrespective of changes in the current amplitude, as if the direction and amplitude of the unitary current regulates the open time. This is supported by further observations that replacement of extracellular Cl - with gluconate - diminishes the inward tail current (Cl - efflux) at a membrane potential of -100 mV due to the lowered outward current (gluconate - influx) at membrane potential of 100 mV. These results suggest that the direction and rate of charge-carrier movement regulate the open time of mPanx1, and that the previously reported voltage-dependence of Panx1 channel gating is not directly mediated by the membrane potential but rather by the direction and amplitude of currents through the channel.

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 2 -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 2+ concentration ([Ca 2+ ] i ) caused by switching to an EGTA-containing Ca 2+ -free solution from normal one elevated the procaterol-induced increasing rate of CBF. These observations suggest that Ca 2+ -dependent PDE1 controls cAMP-stimulated CBF increase. Either application of 8MmIBMX (8-methoxymethyl-IBMX, a selective PDE1 inhibitor), BAPTA-AM (an intracellular Ca 2+ 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.

The typical response to the lowering of plasma Na + concentration and blood pressure in our body involves the release of aldosterone from the adrenal glands, which triggers the reabsorption of sodium in the kidney. Although the effects of aldosterone on this physiological mechanism were extensively studied in the past decades, there are still some aspects to be fully elucidated. In the present study, we propose for the first time a new approach based on Raman spectroscopy to monitor the ionic activity in aldosterone-treated A6 renal epithelial cells. This spectroscopic technique is capable of probing the cells through their thickness in a non-destructive and nimble way. The spectroscopic variations of the Raman bands associated to the O-H stretching of water were correlated to the variations of ionic concentration in the intracellular and extracellular fluids. The increase of Na + concentration gradients was clearly visualized in the cytosol of aldosterone-treated cells. The enhancement of the Na + current density induced by aldosterone was estimated from the variation of the ionic chemical potential across the intracellular space. In addition, the variation of the O-H Raman bands of water was used to quantify the cell thickness, which was not affected by aldosterone.

Down syndrome is a leading cause of congenital intellectual disability caused by an additional copy of the chromosome 21. Patients display physiological and morphological changes affecting the brain and its function. Previously we showed that Ts1Cje and Ts2Cje, Down syndrome mouse models carrying overlapping trisomic segments of different length, show similar ventriculomegaly and neurogenesis dysfunction leading to the hypothesis of a cause-consequence relationship between these phenotypes. However, we here discovered that Ts1Rhr Down syndrome model, carrying an even shorter trisomic segment, was sufficient to trigger ventricular enlargement and ependymal cilia beating deficiency without affecting neurogenesis. We further found that Pcp4 gene on the Ts1Rhr trisomic segment is expressed in ependymal cells, and its resumption to two copies rescued both ventricular enlargement and cilia dysfunction in Ts1Rhr mice. This work underlines a Pcp4-dependent ciliopathy in Down syndrome brain affecting cerebrospinal fluid flow., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background : Hypotonic shock induces cytocidal effects through cell rupture, and cancer therapy based on this mechanism has been clinically administered to hepatocellular carcinoma patients. We herein investigated the effectiveness of hypotonic shock combined with the inhibition of regulatory volume decrease as cancer therapy for hepatocellular carcinoma. Methods : Morphological changes in human hepatocellular carcinoma cell lines were observed under a differential interference contrast microscope connected to a high-speed digital video camera. Cell volume changes under hypotonic shock with or without chloride, potassium, or water channel blockers were observed using a high-resolution flow cytometer. In order to investigate cytocidal effects, the number of surviving cells was compared after exposure to hypotonic solution with and without each channel blocker (re-incubation experiment). Results : Video recordings showed that cells exposed to distilled water rapidly swelled and then ruptured. Cell volume measurements revealed regulatory volume decrease under mild hypotonic shock, whereas severe hypotonic shock increased the number of broken fragments as a result of cell rupture. Moreover, regulatory volume decrease was inhibited in cells treated with each channel blocker. Re-incubation experiments showed the cytocidal effects of hypotonic shock in cells exposed to hypotonic solution, and additional treatments with each channel blocker enhanced these effects. Conclusion : The inhibition of regulatory volume decrease with chloride, potassium, or water channel blockers may enhance the cytocidal effects of hypotonic shock in hepatocellular carcinoma. Hypotonic shock combined with the inhibition of regulatory volume decrease was a more effective therapy than hypotonic shock alone.

A PPARα (peroxisome proliferation activation receptor α) agonist (GW7647) activates nitric oxide synthase 1 (NOS1) to produce NO leading to cGMP accumulation in antral mucous cells. In this study, we examined how PPARα activates NOS1. The NO production stimulated by GW7647 was suppressed by inhibitors of PI3K (wortmannin) and Akt (AKT 1/2 Kinase Inhibitor, AKT-inh), although it was also suppressed by the inhibitors of PPARα (GW6471) and NOS1 (N-PLA). GW7647 enhanced the ACh (acetylcholine)-stimulated exocytosis (Ca(2+)-regulated exocytosis) mediated via NO, which was abolished by GW6471, N-PLA, wortmannin, and AKT-inh. The Western blotting revealed that GW7647 phosphorylates NOS1 via phosphorylation of PI3K/Akt in antral mucous cells. The immunofluorescence examinations demonstrated that PPARα existing with NOS1 co-localizes with PI3K and Akt in the cytoplasm of antral mucous cells. ACh alone and AACOCF3, an analogue of arachidonic acid (AA), induced the NOS1 phosphorylation via PI3K/Akt to produce NO, which was inhibited by GW6471. Since AA is a natural ligand for PPARα, ACh stimulates PPARα probably via AA. In conclusion, PPARα activates NOS1 via PI3K/Akt phosphorylation to produce NO in antral mucous cells during ACh stimulation.

The primary cilium is a hair like structure protruding from most mammalian cells. The basic design of the primary cilium consists of a nine microtubule doublet structure (the axoneme). The Inv compartment, a distinct proximal segment of the ciliary body, is defined as the region in which the Inv protein is localized. Inv gene is a responsible gene for human nephronophthisis type2 (NPHP2). Here, we show that renal cilia have a short proximal microtubule doublet region and a long distal microtubule singlet region. The length of the Inv compartment was similar to that of the microtubule doublet region, suggesting a possibility that the doublet region is the structural basis of the Inv compartment. Respiratory cilia of inv mouse mutants had ciliary rootlet malformation and showed reduced ciliary beating frequency and ciliary beating angle, which may explain recurrent bronchitis in NPHP2 patients. In multiciliated tracheal cells, most Inv proteins were retained in the basal body and did not accumulate in the Inv compartment. These results suggest that the machinery to transport and retain Inv in cilia is different between renal and tracheal cilia and that Inv may function in the basal body of tracheal cells., (© 2015 Wiley Periodicals, Inc.)

Vibrio cholerae colonizes the lumen of the proximal small intestine, which has an alkaline environment, and secretes cholera toxin (CT) through a type II secretion machinery. V. cholerae possesses the intrabacterial nanotransportation system (ibNoTS) for transporting CT from the inner portion toward the peripheral portion of the cytoplasm, and this system is controlled by extrabacterial pH. Association of ATP with ibNoTS has not yet been examined in detail. In this study, we demonstrated by immunoelectron microscopy that ibNoTS of V. cholerae under the extrabacterial alkaline condition was inhibited by ATP inhibitors, 2,4-dinitrophenol (DNP), a protonophore, or 8-amino-adenosine which produces inactive form of ATP. The inhibition of CT transport can be reversed by neutralization of DNP. Those inhibitions were associated with decrease of CT secretion by which ibNoTS followed. We propose that ATP closely associates with V. cholerae ibNoTS for transporting CT.

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.

In antral mucous cells, acetylcholine (ACh, 1 μM) activates Ca(2+)-regulated exocytosis, consisting of a peak in exocytotic events that declines rapidly (initial phase) followed by a second slower decline (late phase) lasting during ACh stimulation. GW7647 [a peroxisome proliferation activation receptor α (PPARα) agonist] enhanced the ACh-stimulated initial phase, and GW6471 (a PPARα antagonist) abolished the GW7647-induced enhancement. However, GW6471 produced the delayed, but transient, increase in the ACh-stimulated late phase, and it also decreased the initial phase and produced the delayed increase in the late phase during stimulation with ACh alone. A similar delayed increase in the ACh-stimulated late phase is induced by an inhibitor of the PKG, Rp8BrPETcGMPS, suggesting that GW6471 inhibits cGMP accumulation. An inhibitor of nitric oxide synthase 1 (NOS1), N(5)-[imino(propylamino)methyl]-L-ornithine hydrochloride (N-PLA), also abolished the GW7647-induced-enhancement of ACh-stimulated initial phase but produced the delayed increase in the late phase. However, in the presence of N-PLA, an NO donor or 8BrcGMP enhanced the ACh-stimulated initial phase and abolished the delayed increase in the late phase. Moreover, GW7647 and ACh stimulated NO production and cGMP accumulation in antral mucosae, which was inhibited by GW6471 or N-PLA. Western blotting and immunohistochemistry revealed that NOS1 and PPARα colocalize in antral mucous cells. In conclusion, during ACh stimulation, a PPARα autocrine mechanism, which accumulates NO via NOS1 leading to cGMP accumulation, modulates the Ca(2+)-regulated exocytosis in antral mucous cells., (Copyright © 2014 the American Physiological Society.)

Levtiracetam (Lev), an inhibitor of SV2A (synaptic vesicle protein A2), affected the ATP-dependent priming of Ca(2+)-regulated exocytosis in antral mucous cells of guinea pig. In antral mucous cells, the Ca(2+)-regulated exocytosis, which is activated by acetylcholine (ACh), consists of an initial peak that declines rapidly (initial phase) followed by a second slower decline (late phase). Dinitrophenol (DNP), which depletes ATP, inhibits the ATP-dependent priming. DNP abolished the initial phase by reducing the number of primed granules, Lev decreased the frequency of initial phase, but not in the presence of DNP. Moreover, 8-bromoguanosine 3'5'-cyclic monophosphate (8BrcGMP) accelerates the ATP-dependent priming. 8BrcGMP enhances the frequency of initial phase by increasing the number of primed granule. Lev added prior to 8BrcGMP addition decreased the frequency of initial phase, but Lev added after 8BrcGMP addition did not. Thus, Lev affected the granules in the process of priming, but it did not affect the granules already primed. Lev did not affect [Ca(2+)]i in unstimulated or ACh-stimulated antral mucous cells. Immunohistochemistry and western blotting demonstrated that SV2A exists in antral mucous cells. The results suggest that SV2A plays an essential role in maintaining the process of ATP-dependent priming in antral mucous cells. In conclusion, Lev decreases the frequency of Ca(2+)-regulated exocytosis the number of primed granules by inhibiting SV2A functions, leading to a decrease in antral mucous cells., (© 2013 Elsevier B.V. All rights reserved.)