The purpose of the present study was to clarify roles of cytosolic chloride ion ( Cl ⁻) in regulation of lysosomal acidification [intra‐lysosomal pH (pH_lys)] and autophagy function in human gastric cancer cell line (MKN28). The MKN28 cells cultured under a low Cl ⁻ condition elevated pH_lys 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 pH_lys 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.

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. 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. 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. 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).

We have reported that pH values of ascites and interstitial fluids around the liver in Otsuka Long-Evans Tokushima Fatty (OLETF) rats are significantly lower than normal pH, 7.40, of mammalian body fluids (Biochem Biophys Res Commun 2013, 432:650), and that this lowered pH of interstitial fluid causes the insulin resistance in diabetic patients by decreasing insulin-binding to its receptors (J Physiol Sci 2013, 63:S199). In the preset study, we tried to measure the interstitial fluid pH in diabetic OLETF rats, since the interstitial fluid pH plays key factors in the brain function from a viewpoint of the binding affinity of neurotransmitters to their receptors.

Sei-hai-to (TJ-90, Qing Fei Tang), a Chinese traditional medicine, increases ciliary beat frequency (CBF) and ciliary bend angle (CBA) mediated via cAMP (3′,5′-cyclic adenosine monophosphate) accumulation modulated by Ca²⁺-activated phosphodiesterase 1 (PDE1A). A high concentration of TJ-90 (≥40 μg/mL) induced two types of CBF increases, a transient increase (an initial increase, followed by a decrease) and a sustained increase without any decline, while it only sustained the CBA increase. Upon inhibiting increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) by 10 μM BAPTA-AM (Ca²⁺-chelator, 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) or Ca²⁺/calmodulin-dependent PDE1 by 8MmIBMX (a selective PDE1 inhibitor), TJ-90 (400 μg/mL) induced only the sustained CBF increase without any transient CBF increase. The two types of the CBF increase (the transient increase and the sustained increase) induced by TJ-90 (≥40 μg/mL) were mimicked by the stimulation with both procaterol (100 pM) and ionomycin (500 nM). Thus, TJ-90 stimulates small increases in the intracellular cAMP concentration ([cAMP]_i) and [Ca²⁺]_i in airway ciliary cells of mice. These small increases in [cAMP]_i and [Ca²⁺]_i cause inducing a transient CBF increase or a sustained CBF increase in an airway ciliary cells, depending on the dominant signal, Ca²⁺-signal, or cAMP-signal.

Diabetes mellitus is a well-known common disease and one of the most serious social problems in the worldwide. Although various types of drugs are developed, the number of patients suffering diabetes mellitus is still increasing. Ninjin’yoeito (NYT) is a formula used in traditional herbal medicines Japanese for improvement recovery from various types of medical disorders. However, the effect of NYT on diabetes mellitus has not yet been investigated. In the present study, we tried to clarify the action of NYT on the blood glucose level in diabetes mellitus model mice. We found that intake of NYT diminished the blood glucose level and insulin intolerance in skeletal muscle in streptozotocin (STZ)-induced diabetic mice. NYT treatment also improved acidification of interstitial fluid in the skeletal muscle in STZ-induced diabetic mice. The expression of sodium-coupled monocarboxylate transporter 1 (SMCT1) was increased in proximal, but not distal, colon of STZ-induced diabetic mice. The present study suggests that NYT is a useful formula to improve the hyperglycemia-induced various disturbance by increasing insulin-sensitivity via elevation of interstitial fluid pH.

Excessive nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS) aggravates acute lung injury (ALI) by producing peroxinitrite. We previously showed that the expression of iNOS and lung injury were suppressed by inhalation of a novel iNOS inhibitor, ONO-1714, in mice with Candida-induced ALI, and that nitric oxide produced by iNOS and apoptosis of epithelial cells were found to have a crucial role in Candida-induced ALI. In the present study, we investigated the effect of NO on the apoptosis of alveolar epithelial cells in Candida-induced ALI. Mice were pretreated by inhalation of ONO-1714 or saline (vehicle control of ONO-1714), and were given an intravenous injection of Candida albicans to induce ALI. After 24 h from injection of Candida albicans, we performed bronchoalveolar lavage and removed lung tissues. We assessed apoptosis on the basis of TUNEL staining and caspase 3 activity. Our results showed that apoptosis was suppressed by inhibition of iNOS-derived NO production by ONO-1714 inhalation. The augmented production of NO increased FasL, TNF-α, and mRNA production of Bax of lung that induced apoptosis of alveolar epithelial cells. Inhibition of iNOS-derived NO production by ONO-1714 inhalation ameliorated Candida-induced ALI and improved survival by suppressing apoptosis of alveolar epithelial cells.