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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.

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.

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%.

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.

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.

The effects of intracellular Cl- concentration ([Cl-]i) on acetylcholine (ACh)-stimulated exocytosis were studied in guinea pig antral mucous cells by video microscopy. ACh activated Ca2+-regulated exocytosis (an initial phase followed by a sustained phase). Bumetanide (20 microM) or a Cl- -free (NO3-) solution enhanced it; in contrast, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, a Cl- channel blocker) decreased it and eliminated the enhancement induced by bumetanide or NO3- solution. ACh and Ca2+ dose-response studies demonstrated that NO3- solution does not shift their dose-response curves, and ATP depletion studies by dinitrophenol or anoxia demonstrated that exposure of NO3- solution prior to ATP depletion induced an enhanced initial phase followed by a sustained phase, whereas exposure of NO3- solution after ATP depletion induced only a sustained phase. Intracellular Ca2+ concentration ([Ca2+]i) measurements showed that bumetanide and NO3- solution enhanced the ACh-stimulated [Ca2+]i increase. Measurements of [Cl-]i revealed that ACh decreases [Cl-]i and that bumetanide and NO3- solution decreased [Cl-]i and enhanced the ACh-evoked [Cl-]i decrease; in contrast, NPPB increased [Cl-]i and inhibited the [Cl-]i decrease induced by ACh, bumetanide, or NO3- solution. These suggest that [Cl-]i modulates [Ca2+]i increase and ATP-dependent priming. In conclusion, a decrease in [Cl-]i accelerates ATP-dependent priming and [Ca2+]i increase, which enhance Ca2+-regulated exocytosis in ACh-stimulated antral mucous cells.

The whole cell patch-clamp technique was used to study ionic conductances in fetal distal lung epithelial (FDLE) cells. In unstimulated FDLE cells, K+ conductances were detected in lowered intracellular Cl- concentration ([Cl-]i, < or = 50 mM). The whole cell currents of FDLE cells were increased by elevation of intracellular Ca2+ concentration ([Ca2+]i) or intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration ([cAMP]i). The elevation of [Ca2+]i activated the K+ currents. The amiloride-blockable whole cell currents were activated by [cAMP]i of 1 mM with [Cl-]i of 20 mM and were more frequently detected in the pipette solution without ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) than with it (0.5 mM). When the [Cl-]i was fixed at 50 or 145 mM, however, the increase in these currents was not detected even with cAMP and without EGTA. The amiloride-blockable currents were detected in both the Na+ and K+ pipette solutions. Thus the increase in amiloride-blockable whole cell currents was due to the activation of nonselective cation channels. In FDLE cells treated with terbutaline, which is a beta 2-adrenergic receptor agonist, or forskolin, these currents were detected in the pipette solution containing 20 mM Cl- but were suppressed with time when the pipette solution contained 50 or 145 mM Cl-. It seems likely that maintenance of [Cl-]i at the lowered level is an important requirement for the FDLE cells to activate the amiloride-blockable whole cell currents. It is proposed that cellular mechanisms, such as cell shrinkage, exist to reduce the [Cl-]i in response to cAMP.

The effect of antidiuretic hormone (ADH) on a distal nephron cell line (A6) was studied using the whole cell patch-clamp technique. A6 cells were cultured on a permeable support filter for 10-14 days in media containing 10% fetal bovine serum without supplemental aldosterone. In the unstimulated condition A6 cells had very small conductances of Na+,K+, and Cl-. Arginine vasotocin (AVT, 140 mU/ml, 280 nM) evoked a "transient" increase in whole cell currents as did dibutyryl-adenosine 3',5'-cyclic monophosphate (5 mM). These transients consisted of two components; one was the nonselective cation conductance, and the other was the Cl- conductance. Activation of these conductances was dependent on intracellular Cl- concentration ([Cl-]i). At low [Cl-]i (< or = 50 mM) both conductances were activated, whereas when [Cl-]i was 80 mM, only the Cl- conductance was activated. At high [Cl-]i (125 mM), both conductances were inhibited. It seems likely that the [Cl-]i maintained at a low level (< or = 50 mM) is an important requirement for A6 cells to respond to AVT.

We studied the regulatory mechanism of Na+ absorption in fetal distal lung epithelium (FDLE), using patch clamp and single cell imaging techniques. A beta 2-agonist activated a 28 pS non-selective cation channel (NSCC) by: 1) producing a dependency of the NSCC activity on the cytosolic [Cl-] ([Cl-]c); 2) inducing a reduction in the [Cl-]c from 45 to 25 mM which directly activated the beta 2-treated NSCC. The beta 2-agonist-induced decrease in [Cl-]c was caused by activation of Ca(2+)-activated K+ channel and cAMP-activated Cl- channel. A development of the [Cl-]c-dependency and a reduction in [Cl-]c act as a second messenger of the beta 2 agonist signal transduction pathway in this Na+ transporting epithelium.

The effect of antidiuretic hormone (ADH) on a distal nephron cell line (A6) was studied using the whole cell patch clamp technique. Arginine vasotocin (AVT, 140mU/ml) evoked a "transient" increase in whole cell currents as did dibutyryl cyclic AMP (5 mM). These transients consisted of two components: one was the non-selective cation conductance and the other was the Cl- conductance. The transient was evoked by AVT in the lower cytosolic Cl- concentration ([Cl-]i) (< approximately 50 mM), and was suppressed by higher concentrations of [Cl-]i (125 mM) in A6 cells. It seems likely that the [Cl-]i maintained at a lower level is an important requirement for A6 cells to respond to AVT.

Intracellular Cl- activity (aiCl) and transmembrane potential (delta psi m) of canine submandibular gland cells were measured in vitro by using Cl- sensitive liquid membrane microelectrodes. Under the conditions of superfusion with a control solution, average Cl- activity of the resting cells was slightly higher than those predicted from Nernstian equilibrium.