Analysis of Aprotinin, a Protease Inhibitor, Action on the Trafficking of Epithelial Na+ Channels (ENaC) in Renal Epithelial Cells Using a Mathematical Model.

BACKGROUND/AIM: Epithelial Na+ channels (ENaC) play a crucial role in control of blood pressure by regulating renal Na+ reabsorption. Intracellular trafficking of ENaC is one of the key regulators of ENaC function, but a quantitative description of intracellular recycling of endogenously expressed ENaC is unavailable. We attempt here to provide a model for intracellular recycling after applying a protease inhibitor under hypotonic conditions. METHODS: We simulated the ENaC-mediated Na+ transport in renal epithelial A6 cells measured as short-circuit currents using a four-state mathematical ENaC trafficking model. RESULTS: We developed a four-state mathematical model of ENaC trafficking in the cytosol of renal epithelial cells that consists of: an insertion state of ENaC that can be trafficked to the apical membrane state (insertion rate); an apical membrane state of ENaC conducting Na+ across the apical membrane; a recycling state containing ENaC that are retrieved from the apical membrane state (endocytotic rate) and then to the insertion state (recycling rate) communicating with the apical membrane state or to a degradation state (degradation rate). We studied the effect of aprotinin (a protease inhibitor) blocking protease-induced cleavage of the extracellular loop of γ ENaC subunit on the rates of intracellular ENaC trafficking using the above-defined four-state mathematical model of ENaC trafficking and the recycling number relative to ENaC staying in the apical membrane. We found that aprotinin significantly reduced the insertion rate of ENaC to the apical membrane by 40%, the recycling rate of ENaC by 81%, the cumulative time of an individual ENaC staying in the apical membrane by 32%, the cumulative life-time after the first endocytosis of ENaC by 25%, and the cumulative Na+ absorption by 31%. The most interesting result of the present study is that cleavage of ENaC affects the intracellular ENaC trafficking rate and determines the residency time of ENaC, indicating that more active cleaved ENaCs stay longer at the apical membrane contributing to transcellular Na+ transport via an increase in recycling of ENaC to the apical membrane. CONCLUSION: The extracellular protease-induced cleavage of the extracellular loop of γ ENaC subunit increases transcellular epithelial Na+ transport by elevating the recycling rate of ENaC due to an increase in the recycling rate of ENaCs associated with increases in the insertion rate of ENaC.

Lowered extracellular pH is involved in the pathogenesis of skeletal muscle insulin resistance.

Insulin resistance in the skeletal muscle is manifested by diminished insulin-stimulated glucose uptake and is a core factor in the pathogenesis of type 2 diabetes mellitus (DM), but the mechanism causing insulin resistance is still unknown. Our recent study has shown that pH of interstitial fluids was lowered in early developmental stage of insulin resistance in OLETF rats, a model of type 2 DM. Therefore, in the present study, we confirmed effects of the extracellular pH on the insulin signaling pathway in a rat skeletal muscle-derived cell line, L6 cell. The phosphorylation level (activation) of the insulin receptor was significantly diminished in low pH media. The phosphorylation level of Akt, which is a downstream target of the insulin signaling pathway, also decreased in low pH media. Moreover, the insulin binding to its receptor was reduced by lowering extracellular pH, while the expression of insulin receptors on the plasma membrane was not affected by the extracellular pH. Finally, insulin-stimulated 2-deoxyglucose uptake in L6 cells was diminished in low pH media. Our present study suggests that lowered extracellular pH conditions may produce the pathogenesis of insulin resistance in skeletal muscle cells.

Improvement of insulin resistance, blood pressure and interstitial pH in early developmental stage of insulin resistance in OLETF rats by intake of propolis extracts.

Propolis, a resinous mixture collected from plants by the Apis mellifera bee, contains high level nutrient factors including vitamins, polyphenols, and amino acids that would be expected to improve insulin sensitivity. Insulin resistance would secondarily cause elevation of blood pressure and increase the risk of cardiovascular diseases. The purpose of this study is to investigate the effect of propolis extracts on blood glucose levels and blood pressures in an early developmental stage of insulin resistance in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. OLETF rats (10 weeks old) were divided into three different groups: normal diet, 0.1% propolis diet, and 0.5% propolis diet. After 8 weeks, blood glucose levels, blood pressures, plasma metabolic factors and hormones, and interstitial fluid pH were measured. Casual blood glucose levels were decreased associated with a reduction of plasma insulin levels in both propolis diet groups compared with normal diet group. Propolis decreased systolic blood pressure with no significant changes in plasma aldosterone levels. We also found that interstitial fluid pH in ascites, liver, and skeletal muscle was higher in rats fed propolis diet than rats fed normal diet. These data suggests that dietary propolis improves insulin sensitivity and blood pressures in the early stage of the process in development of insulin resistance, which may be mediated by suppression of metabolic acidosis.

A regulatory role of K(+)-Cl(-) cotransporter in the cell cycle progression of breast cancer MDA-MB-231 cells.

K(+)-Cl(-) cotransporter (KCC) has been shown to be involved in cell proliferation as well as cell volume regulation. A regulatory role of KCC in cell cycle progression of breast cancer MDA-MB-231 cells was explored by using synchronized MDA-MB-231 cells and dihydro-indenyloxy-alkanoic acid (DIOA), a potent inhibitor of KCC. MDA-MB-231 cells cultured in the presence of DIOA exhibited an increase in cell volume, a decrease in intracellular Cl(-) concentration, and reduction in cell proliferation with the G0/G1 phase arrest, which was accompanied with down-regulation of cyclin D1 and cyclin E2, and up-regulation of p21. Among these molecules, the expression of cyclin E2, a molecule essential for the transition from G1 to S phase, was markedly suppressed by DIOA treatment. DIOA-mediated up- or down-regulation of these molecules occurred at the transcriptional level. These findings suggest that KCC plays an important role in the early phase of cell cycle progression by regulating the expression of cyclin D1, cyclin E2, and p21, the molecules essential for the cell cycle progression.

Therapeutic potential of multifunctional myricetin for treatment of type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by chronic hyperglycemia, insulin resistance, and insufficient insulin secretion. It is considered that chronic hyperglycemia causes serious problems due to diabetic complications such as retinopathy, nephropathy, and neuropathy. Primarily, treatment in T2DM is pharmacologically tried by using drugs that are insulin sensitizers, insulin secretagogues, α-glucosidase inhibitors, and glucose transporter inhibitors. However, long-term application of these drugs frequently induces various harmful side effects, suggesting that the importance of taking advantage of natural products like phytochemicals. Accordingly, flavonoids, a group of phytochemicals, have attracted attention as components of natural products which are effective in the treatment of several diseases containing T2DM and are strongly recommended as food supplements to ameliorate T2DM-related complications. Several well-studied flavonoids such as quercetin and catechin are known to have anti-diabetic, anti-obesity, and anti-hypertensive actions, although a huge number of flavonoids are still under investigation and their actions are not fully understood. In this situation, myricetin is being shown to be a multiple bioactive compound to prevent and/or suppress hyperglycemia through inhibiting digestion and uptake of saccharides and enhancing insulin secretion as a possible GLP-1 receptor agonist, and to ameliorate T2DM-related complications by protecting endothelial cells from oxidative stress induced by hyperglycemia. In this review, we summarize the multiple effects of myricetin on the targets of T2DM treatment, comparing with different flavonoids.

Mumefural prevents insulin resistance and amyloid-beta accumulation in the brain by improving lowered interstitial fluid pH in type 2 diabetes mellitus.

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.

Hypotonic stress upregulates ß- and γ-ENaC expression through suppression of ERK by inducing MKP-1.

We investigated a physiological role for ERK, a member of the MAPK family, in the hypotonic stimulation of epithelial Na(+) channel (ENaC)-mediated Na(+) reabsorption in renal epithelial A6 cells. We show that hypotonic stress causes a major dephosphorylation of ERK following a rapid transient phosphorylation. PD98059 (a MEK inhibitor) increases dephosphorylated ERK and enhances the hypotonic-stress-stimulated Na(+) reabsorption. ERK dephosphorylation is mediated by MAPK phosphatase (MKP). Hypotonic stress activates p38, which in turn induces MKP-1 and to a lesser extent MKP-3 mRNA expression. Inhibition of p38 suppresses MKP-1 induction, preventing hypotonic stress from dephosphorylating ERK. Inhibition of MKP-1 and -3 by the inhibitor NSC95397 also suppresses the hypotonicity-induced dephosphorylation of ERK. NSC95397 reduces both ß- and γ-ENaC mRNA expression and ENaC-mediated Na(+) reabsorption stimulated by hypotonic stress. In contrast, pretreatment with PD98059 significantly enhances mRNA and protein expression of ß- and γ-ENaC even under isotonic conditions. However, PD98059 only stimulates Na(+) reabsorption in response to hypotonic stress, suggesting that ERK inactivation by itself (i.e., under isotonic conditions) is not sufficient to stimulate Na(+) reabsorption, even though ERK inactivation enhances ß- and γ-ENaC expression. Based on these results, we conclude that hypotonic stress stimulates Na(+) reabsorption through at least two signaling pathways: 1) induction of MKP-1 that suppresses ERK activity and induces ß- and γ-ENaC expression, and 2) promotion of translocation of the newly synthesized ENaC to the apical membrane.

An inhibitor of Na(+)/H(+) exchanger (NHE), ethyl-isopropyl amiloride (EIPA), diminishes proliferation of MKN28 human gastric cancer cells by decreasing the cytosolic Cl(-) concentration via DIDS-sensitive pathways.

BACKGROUND/AIMS: Tumor cells produce a large amount of acidic metabolites due to their high metabolic condition. However, cytosolic pH (pH(c)) of tumor cells is identical to or even slightly higher than that of normal cells. To maintain pH(c) at a normal or higher level, tumor cells would have to have higher expression and/or activity of H(+) transporting systems than normal cells. The purpose of the present study was to identify effects of ethyl-isopropyl amiloride (EIPA, an inhibitor of Na(+)/H(+) exchanger (NHE)) on proliferation of human gastric cancer MKN28 cells. METHODS: Effects of EIPA on proliferation, pH(c), [Cl(-)](c) and expression of proteins regulating cell cycle and MAPKs were studied in MKN28 expressing NHE exposed to EIPA for 48 h. RESULTS: EIPA suppressed proliferation of MKN28 cells by causing G(0)/G(1) arrest without any significant effects on pH(c), but associated with reduction of [Cl(-)](c). Although EIPA alone had no effects on pH(c), EIPA co-applied with DIDS (an inhibitor of Cl(-)/HCO(3)(-) exchangers; i.e., anion exchanger (AE) and Na+-driven Cl(-)/HCO(3)(-) exchanger (NDCBE)) reduced pH(c), suggesting that DIDS-sensitive Cl(-)/HCO(3)(-) transporters such as AE and/or NDCBE keep pH(c) normal by stimulating HCO(3)(-) uptake coupled with Cl(-) release under an NHE-inhibited condition. EIPA-induced lowered [Cl(-)](c) up-regulated expression of p21associated with phosphorylation of MAPKs, suppressing proliferation associated with G(0)/G(1) arrest. CONCLUSIONS: EIPA suppressed proliferation of MKN28 cells through up-regulation of p21 expression via reduction of [Cl(-)](c) as a result from DIDS-sensitive Cl(-)/HCO(3)(-) exchanger-mediated compensation for keeping pH(c) normal under an NHE-inhibited condition. This is the first study revealing that an NHE inhibitor suppressed the proliferation of cancer cells by reducing [Cl(-)](c) but not pH(c).

K(+)-Cl(-) cotransporter 1 (KCC1) negatively regulates NGF-induced neurite outgrowth in PC12 cells.

Potassium chloride cotransporters (KCCs) mediate electroneutrally-coupled transport of K(+) and Cl(-), and play crucial roles in various cell functions including regulation of cell volume and homeostasis of cellular Cl(-)content. Four isoforms of KCCs (KCC1, 2, 3, and 4) have been identified. KCC1 is ubiquitously expressed, whereas KCC2 is mainly expressed in neuronal cells of central nervous system. KCC3 is highly expressed in heart, skeletal muscle, kidney, lung and placenta. KCC4 is mainly expressed in epithelial cells. In this study, we investigated roles of KCCs in NGF-induced neurite outgrowth of rat pheochromocytoma PC12 cells. The most abundantly expressed isoform in PC12 cells was KCC1. Inhibition of KCCs using [(dihydronindenyl)oxy] alkanoic acid (DIOA), an inhibitor of KCCs, enhanced the NGF-induced neurite outgrowth of PC12 cells in a dose-dependent manner. Treatment of PC12 cells with NGF significantly decreased mRNA expression of KCC1, whereas other isoforms, KCC2-4, showed no changes in their mRNA expression in response to NGF treatment. Knockdown of KCC1 using small interfering RNA (siRNA) enhanced the NGF-induced neurite outgrowth. These results suggest that KCC1 negatively regulates the NGF-induced neurite outgrowth of PC12 cells.

Enhancement of tubulin polymerization by Cl(-)-induced blockade of intrinsic GTPase.

In growing neurite of neuronal cells, it is suggested that α/ß-tubulin heterodimers assemble to form microtubule, and assembly of microtubule promotes neurite elongation. On the other hand, recent studies reveal importance of intracellular Cl(-) in regulation of various cellular functions such as cell cycle progression, differentiation, cell migration, and elongation of neurite in neuronal cells. In this study, we investigated effects of Cl(-) on in vitro tubulin polymerization. We found that efficiency of in vitro tubulin polymerization (the number of microtubule) was higher (3 to 5-fold) in Cl(-)-containing solutions than that in Cl(-)-free solutions containing Br(-) or NO(3)(-). On the other hand, GTPase activity of tubulin was lower (2/3-fold) in Cl(-)-containing solutions than that in Cl(-)-free solutions containing Br(-) or NO(3)(-). Efficiency of in vitro tubulin polymerization in solutions containing a non-hydrolyzable analogue of GTP (GpCpp) instead of GTP was much higher than that in the presence of GTP. Effects of replacement of GTP with GpCpp on in vitro tubulin polymerization was weaker in Cl(-) solutions (10-fold increases) than that in Br(-) or NO(3)(-) solutions (20-fold increases), although the efficiency of in vitro tubulin polymerization in Cl(-) solutions containing GpCpp was still higher than that in Br(-) or NO(3)(-) solutions containing GpCpp. Our results suggest that a part of stimulatory effects of Cl(-) on in vitro tubulin polymerization is mediated via an inhibitory effect on GTPase activity of tubulin, although Cl(-) would also regulate in vitro tubulin polymerization by factors other than an inhibitory effect on GTPase activity.

Quercetin stimulates NGF-induced neurite outgrowth in PC12 cells via activation of Na(+)/K(+)/2Cl(-) cotransporter.

We have recently reported that Na(+)/K(+)/2Cl(-) cotransporter isoform 1 (NKCC1) plays an essential role in nerve growth factor (NGF)-induced neurite outgrowth in PC12D cells. On the other hand, it has been reported that dietary flavonoids, such as quercetin, apigenin, and luteolin, stimulate various ion transporters. In the present report, we investigated the effect of quercetin, a flavonoid, on NGF-induced neurite outgrowth in PC12 cells (the parental strain of PC12D cells). Quercetin stimulated the NGF-induced neurite outgrowth in a dose-dependent manner. Knockdown of NKCC1 by RNAi methods abolished the stimulatory effect of flavonoid. Quercetin stimulated NKCC1 activity (measured as bumetanide-sensitive (86)Rb influx) without any increase in the expression level of NKCC1 protein. The stimulatory effect of quercetin on neurite outgrowth was dependent upon extracellular Cl(-). These observations indicate that quercetin stimulates the NGF-induced neurite outgrowth via an increase in Cl(-) incorporation into the intracellular space by activating NKCC1 in PC12 cell.

Regulation of epithelial sodium transport via epithelial Na+ channel.

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.

Intracellular chloride regulates cell proliferation through the activation of stress-activated protein kinases in MKN28 human gastric cancer cells.

Recently, we reported that reduction of intracellular Cl(-) concentration ([Cl(-)](i)) inhibited proliferation of MKN28 gastric cancer cells by diminishing the transition rate from G(1) 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 G(1)/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 G(1)/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 G(1) 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.

Chloride ion modulates cell proliferation of human androgen-independent prostatic cancer cell.

In the present study, we investigated if the intracellular Cl(-) affects cell growth and cell cycle progression of androgen-independent prostate cancer PC3 cells. PC3 cells cultured in a medium containing 113 mM Cl(-) for 96 h grew up 9-fold in cell number, while PC3 cells cultured in an 8 mM-Cl(-)-containing culture medium showed complete arrest of cell growth even after culture for 96 h. Exposure of cells to the 8 mM-Cl(-) culture medium diminished phosphorylation levels of Rb and cdc2, which are respectively key accelerators of transition from G(1) to S phase and G(2) to M phase in cell cycle progression. Culturing cells in the 8 mM-Cl(-)-containing culture medium upregulated the protein expression level of p21 (a CDK inhibitor) inhibiting transition of G(1) to S phase, and diminished the incorporation of 5-ethynyl-2'-deoxyuridine (EdU; a thymidine analogue) into DNA. These results suggest that cells cultured in the low Cl(-) medium prolonged the duration of all phases of the cell cycle (G(1), S, and G(2)/M), thereby abolishing overall cell cycle progression. Effects of culturing cells in the low Cl(-) culture medium on cell cycle progression would be mediated via a change in the intracellular Cl(-) concentration ([Cl(-)](i)), since [Cl(-)](i) was decreased under a low Cl(-) culture medium. To clarify this possibility, we studied effects of furosemide and bumetanide, Na+/K+/2Cl(-) cotransporter (NKCC) inhibitors, on proliferation of PC3 cells. Furosemide and bumetanide decreased [Cl(-)](i) and cell growth of PC3 cells. These results suggest that a change in [Cl(-)](i) would play a critical role in this growth mechanism.

Quercetin stimulates Na+/K+/2Cl- cotransport via PTK-dependent mechanisms in human airway epithelium.

We investigated regulatory mechanisms of Cl(-) secretion playing an essential role in the maintenance of surface fluid in human airway epithelial Calu-3 cells. The present study reports that quercetin (a flavonoid) stimulated bumetanide-sensitive Cl(-) secretion with reduction of apical Cl(-) conductance, suggesting that quercetin stimulates Cl(-) secretion by activating an entry step of Cl(-) across the basolateral membrane through Na(+)/K(+)/2Cl(-) cotransporter (NKCC1). To clarify the mechanism stimulating NKCC1 by quercetin, we verified involvement of protein kinase (PK)A, PKC, protein tyrosine kinase (PTK), and cytosolic Ca(2+)-dependent pathways. A PKA inhibitor (PKI-14-22 amide), a PKC inhibitor (Gö 6983) or a Ca(2+) chelating agent did not affect the quercetin-stimulated Cl(-) secretion. On the other hand, a PTK inhibitor (AG18) significantly diminished the stimulatory action of quercetin on Cl(-) secretion without inhibitory effects on apical Cl(-) conductance, suggesting that a PTK-mediated pathway is involved in the stimulatory action of quercetin. The quercetin action on Cl(-) secretion was suppressed with brefeldin A (BFA, an inhibitor of vesicular transport from ER to Golgi), and the BFA-sensitive Cl(-) secretion was not observed in the presence of an epidermal growth factor receptor (EGFR) kinase inhibitor (AG1478), suggesting that quercetin stimulates Cl(-) secretion by causing the EGFR kinase-mediated translocation of NKCC1 or an NKC1-activating factor to the basolateral membrane in human airway epithelial Calu-3 cells. However, the surface density of NKCC1 was not increased by quercetin, but quercetin elevated the activity of NKCC1. These observations indicate that quercetin stimulates Cl(-) secretion by activating NKCC1 via translocation of an NKCC1-activating factor through an EGFR kinase-dependent pathway.

Action of N-acylated ambroxol derivatives on secretion of chloride ions in human airway epithelia.

We report the effects of new N-acylated ambroxol derivatives (TEI-588a, TEI-588b, TEI-589a, TEI-589b, TEI-602a and TEI-602b: a, aromatic amine-acylated derivative; b, aliphatic amine-acylated derivative) induced from ambroxol (a mucolytic agent to treat human lung diseases) on Cl(-) secretion in human submucosal serous Calu-3 cells under a Na(+)/K(+)/2Cl(-) cotransporter-1 (NKCC1)-mediated hyper-secreting condition. TEI-589a, TEI-589b and TEI-602a diminished hyper-secretion of Cl(-) by diminishing the activity of NKCC1 without blockade of apical Cl(-) channel (TEI-589a>TEI-602a>TEI-589b), while any other tested compounds including ambroxol had no effects on Cl(-) secretion. These indicate that the inhibitory action of an aromatic amine-acylated derivative on Cl(-) secretion is stronger that that of an aliphatic amine-acylated derivative, and that 3-(2,5-dimethyl)furoyl group has a strong action in inhibition of Cl(-) secretion than cyclopropanoyl group. We here indicate that TEI-589a, TEI-589b and TEI-602a reduce hyper-secretion to an appropriate level in the airway, providing a possibility that the compound can be an effective drug in airway obstructive diseases including COPD by reducing the airway resistance under a hyper-secreting condition.

Regulation of paracellular Na+ and Cl(-) conductances by hydrostatic pressure.

The effect of hydrostatic pressure on the paracellular ion conductance (Gp) composed of the Na(+) conductance (G(Na)) and the Cl(-) conductance (G(Cl)) has been Investigated. Gp, G(Na) and G(Cl) 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, G(Na) and G(Cl) in a magnitude-dependent manner, while the hydrostatic pressure applied from the apical side diminished the osmotic gradient-induced increase in Gp, G(Na) and G(Cl). How the hydrostatic pressure influences Gp, G(Na) and G(Cl) under an isosmotic condition was also investigated. Gp, G(Na) and G(Cl) 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, G(Na) and G(Cl), while apically applied hydrostatic pressure had little effect on Gp, G(Na) or G(Cl). Taken together, these observations suggest that certain factors controlled by the basolateral osmolality and the basolaterally applied hydrostatic pressure mainly regulate the Gp, G(Na) and G(Cl).

Regulation of the paracellular Na+ and Cl- conductances by the NaCl-generated osmotic gradient in a manner dependent on the direction of osmotic gradients.

In the present study, we investigated the effect of osmolality on the paracellular ion conductance (Gp) composed of the Na(+) conductance (G(Na)) and the Cl(-) conductance (G(Cl)). An osmotic gradient generated by NaCl with relatively apical hypertonicity (NaCl-absorption-direction) induced a large increase in the G(Na) associated with a small increase in the G(Cl), whereas an osmotic gradient generated by NaCl with relatively basolateral hypertonicity (NaCl-secretion-direction) induced small increases in the G(Na) and the G(Cl). These increases in the Gp caused by NaCl-generated osmotic gradients were diminished by the application of sucrose canceling the NaCl-generated osmotic gradient. The osmotic gradient generated by apical [corrected] application of sucrose without any NaCl gradients had little effects on the Gp. However, this apical [corrected] application of sucrose produced a precondition drastically quickening the time course of the action of the NaCl-generated osmotic gradient on the Gp. Further, we found that application of the basolateral hypotonicity generated by reduction of NaCl concentration shifted the localization of claudin-1 to the apical from the lateral [corrected] side. These results indicate that the osmotic gradient regulates the paracellular ion conductive pathway of tight junctions via a mechanism dependent on the direction of NaCl gradients associated with a shift of claudin-1 localization to the apical side in renal A6 epithelial cells.

Chloride ions control the G1/S cell-cycle checkpoint by regulating the expression of p21 through a p53-independent pathway in human gastric cancer cells.

The aim of the present study is to investigate whether the chloride affects cell growth and cell-cycle progression of cancer cells. In human gastric cancer MKN28 cells, the culture in the Cl(-)-replaced medium (replacement of Cl(-) by NO(3)(-)) decreased the intracellular chloride concentration ([Cl(-)](i)) and inhibited cell growth. The inhibition of cell growth was due to cell-cycle arrest at the G(0)/G(1) phase caused by diminution of CDK2 and phosphorylated Rb. The culture of cells in the Cl(-)-replaced medium significantly increased expressions of p21 mRNA and protein without any effects on p53. These observations indicate that chloride ions play important roles in cell-cycle progression by regulating the expression of p21 through a p53-independent pathway in human gastric cancer cells, leading to a novel, unique therapeutic strategy for gastric cancer treatment via control of [Cl(-)](i).

Quercetin is a Useful Medicinal Compound Showing Various Actions Including Control of Blood Pressure, Neurite Elongation and Epithelial Ion Transport.

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.