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New Findings: What is the central question of this study?Arachidonic acid (AA) stimulates NO production in antral mucous cells without any increase in [Ca2+]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 [Ca2+]i. 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 Ca2+‐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 [Ca2+]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 Ca2+‐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 Ca2+‐regulated exocytosis. [ABSTRACT FROM AUTHOR]

Xenin25 has a variety of physiological functions in the Gastrointestinal (GI) tract, including ion transport and motility. However, the motility responses in the colon induced by Xenin25 remain poorly understood. Therefore, the effect of Xenin25 on the spontaneous circular muscle contractions of the rat distal colon was investigated using organ bath chambers and immunohistochemistry. Xenin25 induced the inhibition followed by postinhibitory spontaneous contractions with a higher frequency in the rat distal colon. This inhibitory effect of Xenin25 was significantly suppressed by TTX but not by atropine. The inhibitory time (the duration of inhibition) caused by Xenin25 was shortened by the NTSR1 antagonist SR48692, the NK1R antagonist CP96345, the VPAC2 receptor antagonist PG99‐465, the nitric oxide‐sensitive guanylate‐cyclase inhibitor ODQ, and the Ca2+‐dependent K+ channel blocker apamin. The higher frequency of postinhibitory spontaneous contractions induced by Xenin25 was also attenuated by ODQ and apamin. SP‐, NOS‐, and VIP‐immunoreactive neurons were detected in the myenteric plexus (MP) of the rat distal colon. Small subsets of the SP‐positive neurons were also Calbindin positive. Most of the VIP‐positive neurons were also NOS positive, and small subsets of the NK1R‐positive neurons were also VIP positive. Based on the present results, we propose the following mechanism. Xenin25 activates neuronal NTSR1 on the SP neurons of IPANs, and transmitters from the VIP and apamin‐sensitive NO neurons synergistically inhibit the spontaneous circular muscle contractions via NK1R. Subsequently, the postinhibitory spontaneous contractions are induced by the offset of apamin‐sensitive NO neuron activation via the interstitial cells of Cajal. In addition, Xenin25 also activates the muscular NTSR1 to induce relaxation. Thus, Xenin25 is considered to be an important modulator of post prandial circular muscle contraction of distal colon since the release of Xenin25 from enteroendocrine cells is stimulated by food intake. [ABSTRACT FROM AUTHOR]

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 NO3- 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. [ABSTRACT FROM AUTHOR]

Aims/Introduction: In Japan, an ideal bodyweight (IBW) calculated by 22 × height (m)2 has commonly been used in the planning of medical nutrition therapy (MNT). However, there have been concerns regarding calorie deficits in fulfilling resting energy expenditure (REE) for patients with type 2 diabetes undergoing MNT as defined by 25 kcal/kg IBW/day. The objective of the present study was to measure REE in patients with type 2 diabetes and verify the validity of MNT with 25 kcal/kg IBW/day. Materials and Methods: A retrospective cross‐sectional study was carried out in 52 patients with type 2 diabetes (mean age was 65.9 ± 7.3 years, bodyweight 65.0 ± 11.3 kg, body mass index 24.9 ± 3.8 kg/m2). REE was measured by indirect calorimetry. Results: The mean REE was 1,601.0 ± 253.1 kcal/day. Assuming that all patients strictly observed daily energy intake as 25 kcal/kg IBW/day, 41 of 52 patients (78.9%) did not reach their REE. The greater the bodyweight, the greater the difference between assumed energy intake as 25 kcal/kg IBW and REE. Conclusions: We call attention to the potential risk of total dietary energy intake set to 25 kcal/kg IBW/day. Clinicians should carefully plan MNT to not fall below a patient's REE to prevent sarcopenia and ensure MNT continuity. [ABSTRACT FROM AUTHOR]

New Findings: What is the central question of this study? The ciliary beat frequency (CBF) of the airway is controlled by [Ca2+]i. However, the effects of a reduction in [Ca2+]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 [Ca2+]i directly regulates CBF (direct action) and also indirectly regulates CBF via cAMP accumulation controlled by Ca2+‐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: [Ca2+]i plays crucial roles in the regulation of ciliary beat frequency (CBF) and ciliary bend angle (CBA) of airway cilia. Moreover, Ca2+‐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 [Ca2+]i; the direct action changes CBF mediated via [Ca2+]i, and the indirect action changes CBF mediated via cAMP, the accumulation of which is controlled by PDE1 activity. Upon reducing [Ca2+]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 [Ca2+]i; a slight decrease induced by a nominally Ca2+‐free solution (no cAMP accumulation via PDE1) decreases CBF, and an extreme decrease induced by 50 μ m 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 [Ca2+]i is smaller than the increase in CBF, because no PDE1A exists in the CBA‐regulating metabolon. On the contrary, an increase in [Ca2+]i induced by ionomycin, which decreases cAMP accumulation by PDE1A activation, caused a slower procaterol‐stimulated increase in CBF than that decreased by a Ca2+‐free solution. A decrease in [Ca2+]i stimulates cAMP accumulation, whereas an increase in [Ca2+]i inhibits cAMP accumulation in airway ciliary cells. Thus, changes in [Ca2+]i modulate CBF and CBA via cAMP accumulation by controlling the activity of PDE1. [ABSTRACT FROM AUTHOR]

Key points Calcium homeostasis modulator 1 (CALHM1), a new voltage-gated ATP- and Ca2+-permeable channel, plays important physiological roles in taste perception and memory formation., Regulatory mechanisms of CALHM1 remain unexplored, although the biophysical disparity between CALHM1 gating in vivo and in vitro suggests that there are undiscovered regulatory mechanisms., Here we report that CALHM1 gating and association with lipid microdomains are post-translationally regulated through the process of protein S-palmitoylation, a reversible attachment of palmitate to cysteine residues., Our data also establish cysteine residues and enzymes responsible for CALHM1 palmitoylation., CALHM1 regulation by palmitoylation provides new mechanistic insights into fine-tuning of CALHM1 gating in vivo and suggests a potential layer of regulation in taste and memory., Abstract Emerging roles of CALHM1, a recently discovered voltage-gated ion channel, include purinergic neurotransmission of tastes in taste buds and memory formation in the brain, highlighting its physiological importance. However, the regulatory mechanisms of the CALHM1 channel remain entirely unexplored, hindering full understanding of its contribution in vivo. The different gating properties of CALHM1 in vivo and in vitro suggest undiscovered regulatory mechanisms. Here, in searching for post-translational regulatory mechanisms, we discovered the regulation of CALHM1 gating and association with lipid microdomains via protein S-palmitoylation, the only reversible lipid modification of proteins on cysteine residues. CALHM1 is palmitoylated at two intracellular cysteines located in the juxtamembrane regions of the third and fourth transmembrane domains. Enzymes that catalyse CALHM1 palmitoylation were identified by screening 23 members of the DHHC protein acyltransferase family. Epitope tagging of endogenous CALHM1 proteins in mice revealed that CALHM1 is basally palmitoylated in taste buds in vivo. Functionally, palmitoylation downregulates CALHM1 without effects on its synthesis, degradation and cell surface expression. Mutation of the palmitoylation sites has a profound impact on CALHM1 gating, shifting the conductance-voltage relationship to more negative voltages and accelerating the activation kinetics. The same mutation also reduces CALHM1 association with detergent-resistant membranes. Our results comprehensively uncover a post-translational regulation of the voltage-dependent gating of CALHM1 by palmitoylation. [ABSTRACT FROM AUTHOR]

Flavonoids keep us healthy by controlling various body and cellular functions. It is well known that cations, such as Na+, K+, and Ca2+, play important roles in the regulation of body and cellular functions, including generation of action potentials and the resting membrane potential of neural and muscle cells and signal transduction as intracellular second messengers. However, we have little information on the physiological roles of anions, particularly Cl-, in body and cellular functions. Quercetin, a flavonoid, stimulates Na+-K+-2Cl- cotransporter 1 (NKCC1), which is one of the most important ion transporters regulating the cytosolic Cl- concentration ([Cl-]c). Here, we introduce the molecular mechanism by which flavonoids, specifically quercetin, act through elevation of [Cl-]c via activation of NKCC1 on important factors controlling various body and cellular functions, such as (1) antihypertensive actions controlling blood volume dependent on the amounts of renal Na+ reabsorption via expression of the epithelial Na+ channel, (2) neurite-elongating actions via polymerization of tubulin by inhibiting GTPase activity, and (3) antibacterial and antiviral infective actions through stimulation of epithelial Cl- secretion by increasing the driving force for epithelial Cl- secretion. [ABSTRACT FROM AUTHOR]

The purpose of the present study was to clarify roles of cytosolic chloride ion ( Cl−) in regulation of lysosomal acidification [intra-lysosomal pH (pHlys)] and autophagy function in human gastric cancer cell line ( MKN28). The MKN28 cells cultured under a low Cl− condition elevated pHlys and reduced the intra-lysosomal Cl− concentration ([ Cl−]lys) via reduction of cytosolic Cl− concentration ([ Cl−]c), showing abnormal accumulation of LC3II and p62 participating in autophagy function (dysfunction of autophagy) accompanied by inhibition of cell proliferation via G0/G1 arrest without induction of apoptosis. We also studied effects of direct modification of H+ transport on lysosomal acidification and autophagy. Application of bafilomycin A1 (an inhibitor of V-type H+- ATPase) or ethyl isopropyl amiloride [ EIPA; an inhibitor of Na+/ H+ exchanger ( NHE)] elevated pHlys and decreased [ Cl−]lys associated with inhibition of cell proliferation via induction of G0/G1 arrest similar to the culture under a low Cl− condition. However, unlike low Cl− condition, application of the compound, bafilomycin A1 or EIPA, induced apoptosis associated with increases in caspase 3 and 9 without large reduction in [ Cl−]c compared with low Cl− condition. These observations suggest that the lowered [ Cl−]c primarily causes dysfunction of autophagy without apoptosis via dysfunction of lysosome induced by disturbance of intra-lysosomal acidification. This is the first study showing that cytosolic Cl− is a key factor of lysosome acidification and autophagy. [ABSTRACT FROM AUTHOR]

Renal epithelial Na+ transport plays an important role in homeostasis of our body fluid content and blood pressure. Further, the Na+ transport in alveolar epithelial cells essentially controls the amount of alveolar fluid that should be kept at an appropriate level for normal gas exchange. The epithelial Na+ transport is generally mediated through two steps: (1) the entry step of Na+ via epithelial Na+ channel (ENaC) at the apical membrane and (2) the extrusion step of Na+ via the Na+, K+-ATPase at the basolateral membrane. In general, the Na+ entry via ENaC is the rate-limiting step. Therefore, the regulation of ENaC plays an essential role in control of blood pressure and normal gas exchange. In this paper, we discuss two major factors in ENaC regulation: (1) activity of individual ENaC and (2) number of ENaC located at the apical membrane. [ABSTRACT FROM AUTHOR]

Indomethacin (IDM, 10 μm), not aspirin (ASA; 10 μm), enhanced the Ca2+-regulated exocytosis stimulated by 1 μm acetylcholine (ACh) in guinea-pig antral mucous cells. Indomethacin inhibits prostaglandin G/H (PGG/H) and 15 R-hydroperoxy-eicosatetraenoic acid (15R-HPETE) production from arachidonic acid (AA), while ASA inhibits PGG/H production but accelerates 15R-HPETE production. This suggests that IDM accumulates AA. Arachidonic acid (2 μm) enhanced Ca2+-regulated exocytosis in antral mucous cells to a similar extent to IDM. Moreover, a stable analogue of AA, arachidonyltrifluoromethyl ketone (AACOCF3), also enhanced Ca2+-regulated exocytosis, indicating that AA, not products from AA, enhances Ca2+-regulated exocytosis. We hypothesized that AA activates peroxisome proliferation activation receptor α (PPARα), because AA is a natural ligand for PPARα. A PPARα agonist (WY14643; 1 μm) enhanced Ca2+-regulated exocytosis, and a PPARα blocker (MK886; 50 μm) abolished the enhancement of Ca2+-regulated exocytosis induced by AA, IDM, AACOCF3 and WY14643. Western blotting and immunohistochemical examinations demonstrated that PPARα exists in antral mucous cells. Moreover, MK886 decreased the frequency of Ca2+-regulated exocytosis activated by 1 μm ACh or 2 μm thapsigargin alone by 25–30%. Thus, ACh stimulates AA accumulation via an [Ca2+]i increase, which activates PPARα, leading to enhancement of Ca2+-regulated exocytosis in antral mucous cells. A novel autocrine mechanism mediated via PPARα enhances Ca2+-regulated exocytosis in guinea-pig antral mucous cells. [ABSTRACT FROM AUTHOR]

Recently, we reported that reduction of intracellular Cl- concentration ([Cl-]i) inhibited proliferation of MKN28 gastric cancer cells by diminishing the transition rate from G1 to S cell-cycle phase through upregulation of p21, cyclin-dependent kinase inhibitor, in a p53-independent manner. However, it is still unknown how intracellular Cl- regulates p21 expression level. In this study, we demonstrate that mitogen-activated protein kinases (MAPKs) are involved in the p21 upregulation and cell-cycle arrest induced by reduction of [Cl-]i. Culture of MKN28 cells in a low Cl- medium significantly induced phosphorylation (activation) of MAPKs (ERK, p38, and JNK) and G1/S cell-cycle arrest. To clarify the involvement of MAPKs in p21 upregulation and cell growth inhibition in the low Cl- medium, we studied effects of specific MAPKs inhibitors on p21 upregulation and G1/S cell-cycle arrest in MKN28 cells. Treatment with an inhibitor of p38 or JNK significantly suppressed p21 upregulation caused by culture in a low Cl- medium and rescued MKN28 cells from the low Cl--induced G1 cell-cycle arrest, whereas treatment with an ERK inhibitor had no significant effect on p21 expression or the growth of MKN28 cells in the low Cl- medium. These results strongly suggest that the intracellular Cl- affects the cell proliferation via activation of p38 and/or JNK cascades through upregulation of the cyclin-dependent kinase inhibitor (p21) in a p53-independent manner in MKN28 cells. J. Cell. Physiol. 223:764–770, 2010. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Abstract: The effect of hydrostatic pressure on the paracellular ion conductance (Gp) composed of the Na+ conductance (GNa) and the Cl− conductance (GCl) has been Investigated. Gp, GNa and GCl were time-dependently increased after applying an osmotic gradient generated by NaCl with basolateral hypotonicity. Hydrostatic pressure (1–4cm H2O) applied from the basolateral side enhanced the osmotic gradient-induced increase in Gp, GNa and GCl in a magnitude-dependent manner, while the hydrostatic pressure applied from the apical side diminished the osmotic gradient-induced increase in Gp, GNa and GCl. How the hydrostatic pressure influences Gp, GNa and GCl under an isosmotic condition was also investigated. Gp, GNa and GCl were stably constant under a condition with basolateral application of sucrose canceling the NaCl-generated osmotic gradient (an isotonic condition). Even under this stable condition, the basolaterally applied hydrostatic pressure drastically elevated Gp, GNa and GCl, while apically applied hydrostatic pressure had little effect on Gp, GNa or GCl. Taken together, these observations suggest that certain factors controlled by the basolateral osmolality and the basolaterally applied hydrostatic pressure mainly regulate the Gp, GNa and GCl. [Copyright &y& Elsevier]

Abstract: Epithelial sodium channel (ENaC) plays a crucial role in controlling sodium reabsorption in the kidney keeping the normal blood pressure. We previously reported that the expression of ENaC mRNA in the kidney of Dahl salt-sensitive (DS) rats was abnormally regulated by aldosterone, however it is unknown if dietary sodium affects the expression of ENaC and serum and glucocorticoid-regulated kinase 1 (SGK1), which plays an important role in ENaC activation, in DS rats. In the present study, we investigated whether dietary sodium abnormally affects the expression of ENaC and SGK1 mRNA in DS rats. DS and Dahl salt-resistant (DR) rats (8weeks old) were divided into three different groups, respectively: (1) low sodium diet (0.005% NaCl), (2) normal sodium diet (0.3% NaCl), and (3) high sodium diet (8% NaCl). The high sodium diet for 4weeks in DS rats elevated the systolic blood pressure, but did not in any other groups. The expression of α-ENaC mRNA in DS rats was abnormally increased by high sodium diet in contrast to DR rats, while it was normally increased by low sodium diet in DS rats similar to DR rats. The expression of β- and γ-ENaC mRNA in DS rats was also abnormally increased by high sodium diet unlike DR rats. The expression of SGK1 mRNA was elevated by high sodium diet in DS rats, but it was decreased in DR rats. These observations indicate that the expression of ENaC and SGK1 mRNA is abnormally regulated by dietary sodium in salt-sensitively hypertensive rats, and that this abnormal expression would be one of the factors causing salt-sensitive hypertension. [Copyright &y& Elsevier]

1 Lignocaine suppresses insulin-stimulated glucose transport into the cells and insulin-stimulated glycogenesis at doses equivalent to that used in the treatment of muscle pain disorder. We evaluated the direct effect of lignocaine on insulin receptor (IR) kinase activity. 2 After lignocaine (40 mM. approximately equivalent to 1%) or an equal volume (100&mul;) saline had been injected into the tibialis anterior muscle of rat. insulin (50 mM g[sup-1] body weight) was administered into the portal vein in vivo. Immunoprecipitation and immunoblotting were used to detect insulin-mediated tyrosine phosphorylation of both IR-β and insulin receptor substrate (IRS)-I. and insulin-stimulated binding of IRS-1 to p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-K) in the extracted muscle. 3 In the in vitro study, purified IR from rat liver and or recombinant IRS-1 protein with adenosine triphosphate were incubated with lignocaine (4 or 40 mM). 4 Lignocaine reduced insulin-stimulated tyrosine phosphorylation of IR-β to 12.6±5.7% (P<0.001). and IRS-1 to 32.1±18.8% (P<0.01), and also reduced insulin-stimulated binding of IRS-1 to p85 to 27. 4±12.7% (P<0.001) relative to control (100%) in muscle in vivo. 5 The in vitro study revealed that lignocaine directly inhibited both basal and insulin-stimulated tyrosine phosphorylation of IR. 6 These results indicate that clinically used doses of lignocaine inhibit insulin signalling in skeletal muscle. The inhibitory effect of lignocaine on tyrosine kinase activity of the IR underlies the suppression of insulin signalling with lignocaine. [ABSTRACT FROM AUTHOR]

Osmolality has various actions on epithelial transports. To study the effects of osmolality on paracellular ion conductance (Gp), we established a method estimating Gp by measuring the transepithelial potential (Vt) with abolishment of transcellular ion conductance using a modified Ussing chamber in renal epithelia. Gp changed proportionally to the ionic strength of bathing solutions and was independent of Vt. Based on these characteristics, we estimated the paracellular ion conductances of Na+ (GNa) and Cl- (GCI) using equilibrium potentials for Na+ and CI- between apical and basolateral solutions. Secretory directed water flux generated by osmotic gradients associated with NaCl gradients between apical and basolateral solutions increased the Na+-selective Gp. Secretory directed water flux without NaCl gradients itself had little effects on Gp, but produced precondition generating Gp elevation observed with NaCl gradients. In contrast, absorptive directed water flux increased Gp with no discrimination between GNa and GCl. These observations indicate that Gp is composed of Na+ selective and nonselective components, which are selectively regulated by the water fluxes in a direction-dependent manner. [ABSTRACT FROM AUTHOR]

Serum- and glucocorticoid-inducible kinase 1 (SGK1) plays a key role in body NaCl homeostasis by fine tuning of renal NaCl reabsorption in the distal nephron via modulation of epithelial Na+ channel (ENaC) activity, transcription, and trafficking. Although induction of SGK1 is involved in the stimulatory action of hypotonicity on Na+ transport, it is unknown how hypotonicity regulates SGK1 expression. In the present study, we studied if Ca2+ signal is involved in the hypotonic action on SGK1 expression and Na+ transport. Using QRT-PCR and western blotting, we observed that BAPTA/AM (an intracellular Ca2+ chelator) and W7 (a calmodulin antagonist) blunted the hypotonic induction of SGK1 mRNA and protein. Ionomycin (a Ca2+ ionophore) stimulated SGK1 transcription under an isotonic condition. BAPTA/AM retarded the hypotonicity-induced Na+ current. Further, our observation suggests that hypotonicity-induced expression and activity of SGK1 would be associated with expression of αENaC, but not β or γENaC. Taken together, we conclude that hypotonic stress has genomic action on SGK1 mRNA expression in a Ca2+/calmodulin-dependent manner, stimulating Na+ transport and αENaC expression. [ABSTRACT FROM AUTHOR]

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.)

Indomethacin (IDM, 10 microm), not aspirin (ASA; 10 microm), enhanced the Ca(2+)-regulated exocytosis stimulated by 1 microm acetylcholine (ACh) in guinea-pig antral mucous cells. Indomethacin inhibits prostaglandin G/H (PGG/H) and 15R-hydroperoxy-eicosatetraenoic acid (15R-HPETE) production from arachidonic acid (AA), while ASA inhibits PGG/H production but accelerates 15R-HPETE production. This suggests that IDM accumulates AA. Arachidonic acid (2 microm) enhanced Ca(2+)-regulated exocytosis in antral mucous cells to a similar extent to IDM. Moreover, a stable analogue of AA, arachidonyltrifluoromethyl ketone (AACOCF(3)), also enhanced Ca(2+)-regulated exocytosis, indicating that AA, not products from AA, enhances Ca(2+)-regulated exocytosis. We hypothesized that AA activates peroxisome proliferation activation receptor alpha (PPARalpha), because AA is a natural ligand for PPARalpha. A PPARalpha agonist (WY14643; 1 microm) enhanced Ca(2+)-regulated exocytosis, and a PPARalpha blocker (MK886; 50 microm) abolished the enhancement of Ca(2+)-regulated exocytosis induced by AA, IDM, AACOCF(3) and WY14643. Western blotting and immunohistochemical examinations demonstrated that PPARalpha exists in antral mucous cells. Moreover, MK886 decreased the frequency of Ca(2+)-regulated exocytosis activated by 1 microm ACh or 2 microm thapsigargin alone by 25-30%. Thus, ACh stimulates AA accumulation via an [Ca(2+)](i) increase, which activates PPARalpha, leading to enhancement of Ca(2+)-regulated exocytosis in antral mucous cells. A novel autocrine mechanism mediated via PPARalpha enhances Ca(2+)-regulated exocytosis in guinea-pig antral mucous cells.

Maintaining the extracellular K(+) concentration ([K(+)](o)) between 15 and 60 mM induced oscillations in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in rat submandibular acinar cells during stimulation with acetylcholine (ACh, 1 micro M). These [Ca(2+)](i) oscillations were also induced by 1 micro M thapsigargin and were inhibited by 50 micro M La(3+), 1 micro M Gd(3+), or the removal of extracellular Ca(2+), indicating that the [Ca(2+)](i) oscillations were generated by store-operated Ca(2+) entry (SOC). The frequency of the ACh-evoked [Ca(2+)](i) oscillations increased from 0.8 to 2.3 mHz as [K(+)](o) was increased from 15 to 50 mM. TEA (an inhibitor of K(+) channels) also induced [Ca(2+)](i) oscillations at [K(+)](o) of 4.5 or 7.5 mM in ACh-stimulated cells. These data suggest that depolarization causes [Ca(2+)](i) to oscillate in ACh-stimulated submandibular acinar cells. Pertussis toxin (PTX, an inhibitor of G proteins) caused [Ca(2+)](i) to be sustained at a high level in ACh-stimulated cells at 25 mM or 60 mM [K(+)](o). This suggests that the [Ca(2+)](i) oscillations are generated by a periodic inactivation of the SOC channels via PTX-sensitive G proteins, which are stimulated by depolarization. Moreover, in the presence of DBcAMP or forskolin which accumulated cAMP the frequency of the [Ca(2+)](i) oscillations remained constant (approximately 1.2 mHz) when [K(+)](o) was maintained in the range 25-60 mM. Based on these observations in ACh-stimulated submandibular acinar cells, we conclude that depolarization stimulates the PTX-sensitive G proteins, which inactivate the SOC channels periodically ([Ca(2+)](i) oscillation), while hyperpolarization or PTX inhibits the G proteins, maintaining the activation of the SOC channels. Accumulation of cAMP is likely to modulate the PTX-sensitive G proteins.