Ken-ichi Nakajima

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 86Rb 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.

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 G₀/G₁ 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^-/HCO₃^- exchangers; i.e., anion exchanger (AE) and Na+-driven Cl^-/HCO₃^- exchanger (NDCBE)) reduced pH_c, suggesting that DIDS-sensitive Cl^-/HCO₃^- transporters such as AE and/or NDCBE keep pH_c normal by stimulating HCO₃^- 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 G₀/G₁ 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^-/HCO₃^- 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.

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.

NaCl flux between apical and basolateral side recruits claudin-1 to tight junction strands and regulates paracellular transport.

In multicellular organisms, epithelia separate and divide the internal environment maintaining appropriate conditions in each compartment. To maintain homeostasis in these compartments, claudins, major cell adhesion molecules in tight junctions (TJs), regulate movements of several substances through the paracellular pathway (barrier function). In this study, we investigated effects of the flux of several substances between apical and basolateral side on paracellular transport and TJ protein localization. NaCl flux from apical to basolateral side increased paracellular conductance (Gp) and recruited claudin-1 from lateral cell membrane to the apical end with the colocalization with occludin, one of the TJ proteins concentrated at TJ strands. Oppositely-directed flux of sucrose against NaCl flux inhibited these reactions and same directional flux of sucrose with NaCl enhanced the increase of Gp, whereas 10-kDa dextran inhibited these reactions regardless of the side of administration. Our present findings indicated that TJ protein localization and barrier function are regulated depending on the environmental differences between apical and basolateral side.

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

In growing neurite of neuronal cells, it is suggested that α/β-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.

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.

Hydrostatic pressure regulates tight junctions, actin cytoskeleton and transcellular ion transport

In the epithelia and endothelia, tight junctions regulate the movement of several substances through the paracellular pathway, maintaining several gradients between apical and basal compartments including osmolality and hydrostatic pressure. In this study, we show that the change of hydrostatic pressure gradient affected tight junctions as well as actin cytoskeleton, cell height and transcellular ion transport. Hydrostatic pressure gradient from basolateral to apical side increased transepithelial conductance and altered claudin-1 localization within several tens of minutes. These changes were promptly restored by the elimination of hydrostatic pressure gradient. Hydrostatic pressure gradient also induced dynamic changes in the actin structure and cell height. We further found that hydrostatic pressure gradient from basolateral to apical side stimulates transcellular Cl(-) transport. Our present findings indicate that the epithelial cell structures and functions are regulated by the hydrostatic pressure gradient which is generated and maintained by the epithelia themselves.

Intracellular chloride ion concentration in differentiating neuronal cell and its role in growing neurite

Chloride ion (Cl-) is one of the most abundant anions in our body. Increasing evidence suggests that Cl- plays fundamental roles in various cellular functions. We have previously reported that electroneutral cation-chloride cotransporters, such as Na+-K+-2Cl- cotransporter 1 (NKCC1) and K+-Cl- cotransporter 1 (KCC1), are involved in neurite outgrowth during neuronal differentiation. In the present study, we studied if there is correlation between intracellular Cl- concentrations ([Cl-]i) and the length of growing neurites. We measured [Cl-]i in the cell body and growing neurite tips using halide-sensitive fluorescent dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE), revealing that [Cl-]i in the tip of growing neurite was higher than that in cell body in a single cell. Importantly, there was a significant positive correlation between the length of growing neurite and [Cl-]i in neurite tip. Bumtanide (BMT), an inhibitor of NKCC1, significantly inhibited neurite outgrowth and decreased [Cl-]i in neurite tip. The results obtained in the present study and our previous studies together strongly suggest that high [Cl-]i in neurite tip region is crucial for efficient neurite outgrowth.

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 (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 (1–4 cm 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.