Chaperone stress 70 protein (STCH) binds and regulates two acid/base transporters NBCe1-B and NHE1.

Regulation of intracellular pH is critical for the maintenance of cell homeostasis in response to stress. We used yeast two-hybrid screening to identify novel interacting partners of the pH-regulating transporter NBCe1-B. We identified Hsp70-like stress 70 protein chaperone (STCH) as interacting with NBCe1-B at the N-terminal (amino acids 96-440) region. Co-injection of STCH and NBCe1-B cRNA into Xenopus oocytes significantly increased surface expression of NBCe1-B and enhanced bicarbonate conductance compared with NBCe1-B cRNA alone. STCH siRNA decreased the rate of Na(+)-dependent pHi recovery from NH4(+) pulse-induced acidification in an HSG (human submandibular gland ductal) cell line. We observed that in addition to NBCe1-B, Na(+)/H(+) exchanger (NHE)-dependent pHi recovery was also impaired by STCH siRNA and further confirmed the interaction of STCH with NHE1 but not plasma membrane Ca(2+) ATPase. Both NBCe1-B and NHE1 interactions were dependent on a specific 45-amino acid region of STCH. In conclusion, we identify a novel role of STCH in the regulation of pHi through site-specific interactions with NBCe1-B and NHE1 and subsequent modulation of membrane transporter expression. We propose STCH may play a role in pHi regulation at times of cellular stress by enhancing the recovery from intracellular acidification.

Intracellular acidification is associated with changes in free cytosolic calcium and inhibition of action potentials in rat trigeminal ganglion.

The effect of intracellular acidification and subsequent pH recovery in sensory neurons has not been well characterized. We have studied the mechanisms underlying Ca(2+)-induced acidification and subsequent recovery of intracellular pH (pH(i)) in rat trigeminal ganglion neurons and report their effects on neuronal excitability. Glutamate (500 µM) and capsaicin (1 µM) increased intracellular Ca(2+) concentration ([Ca(2+)](i)) with a following decrease in pH(i). The recovery of [Ca(2+)](i) to the prestimulus level was inhibited by LaCl(3) (1 mM) and o-vanadate (10 mM), a plasma membrane Ca(2+)/ATPase (PMCA) inhibitor. Removal of extracellular Ca(2+) also completely inhibited the acidification induced by capsaicin. TRPV1 was expressed only in small and medium sized trigeminal ganglion neurons. mRNAs for Na(+)/H(+) exchanger type 1 (NHE1), pancreatic Na(+)-HCO(3)(-) cotransporter type 1 (pNBC1), NBC3, NBC4, and PMCA types 1-3 were detected by RT-PCR. pH(i) recovery was significantly inhibited by pretreatment with NHE1 or pNBC1 siRNA. We found that the frequency of action potentials (APs) was dependent on pH(i). Application of the NHE1 inhibitor 5'-(N-ethyl-N-isopropyl) amiloride (5 µM) or the pNBC1 inhibitor 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid (500 µM) delayed pH(i) recovery and decreased AP frequency. Simultaneous application of 5'-(N-ethyl-N-isopropyl) amiloride and 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid almost completely inhibited APs. In summary, our results demonstrate that the rise in [Ca(2+)](i) in sensory neurons by glutamate and capsaicin causes intracellular acidification by activation of PMCA type 3, that the pH(i) recovery from acidification is mediated by membrane transporters NHE1 and pNBC1 specifically, and that the activity of these transporters has direct consequences for neuronal excitability.

Anticancer effects of CKD-602 (Camtobell®) via G2/M phase arrest in oral squamous cell carcinoma cell lines.

CKD-602 (7-[2-(N-isopropylamino) ethyl]-(20S)-camptothecin, belotecan) is a synthetic water-soluble camptothecin derivative and topoisomerase I inhibitor that has been shown to exert a clinical anticancer effect on various types of tumor. In the present study, the anticancer effects of CKD-602 on the following three human oral squamous cell carcinoma (OSCC) cell lines originating from Korean cancer patients: YD-8 (tongue), YD-9 (buccal mucosa) and YD-38 (lower gingiva) were analyzed. The apoptotic proportion of the cells and cell cycle position were analyzed using flow cytometry. The expression of cell cycle regulatory proteins was detected by western blot analysis. CKD-602 was demonstrated to exert a time- and dose-dependent antiproliferative effect in all cell lines in vitro, however, susceptibility to CKD-602 at 72 h following treatment varied among the three cell lines, with 50% inhibition of cell viability at concentrations of 2.4 µg/ml for YD-8, 0.18 µg/ml for YD-9 and 0.05 µg/ml for YD-38. To investigate the underlying mechanism of the CKD-602 antiproliferative effect, a cell cycle-analysis was conducted in the three OSCC cell lines and CKD-602 treatment was observed to induce G2/M phase arrest. Furthermore, western blot analysis revealed that the expression levels of phospho-cdc2 (Tyr 15), cyclin A2 and cyclin B1 were increased in a time-dependent manner, following the administration of CKD-602. In the fluorescence-activated cell sorting analysis, the number of apoptotic cells was also increased in a dose-dependent manner following CKD-602 treatment of the OSCC cell lines. The results suggest that CKD-602 may inhibit the proliferation of OSCC oral cancer cells derived from samples from Korean patients by apoptosis and by G2/M phase arrest.

Effects of resveratrol-related hydroxystilbenes on the nitric oxide production in macrophage cells: structural requirements and mechanism of action.

NF-kappaB that plays an important role in iNOS expression is one of the targets of various potential anti-inflammatory agents including resveratrol. Resveratrol contains a structural similarity with estrogen, and there has been speculation about resveratrol as estrogen agonist. In this study, the mechanism and structural requirements of resveratrol and related hydroxystilbenes for the inhibition of LPS-induced nitric oxide production were studied in macrophage cells (RAW 264.7 and J774) by comparing its effect on LPS-induced NF-kappaB translocation and nitric oxide production, and by considering the possibility of involvement of an estrogen receptor. LPS-induced nitric oxide production was inhibited only when cells were treated with resveratrol prior to stimulation with LPS, suggesting that resveratrol does not affect the enzyme itself. A higher concentration of resveratrol than needed for the inhibition of nitric oxide production was required for the inhibition of NF-kappaB mobilization or iNOS expression. Estrogen and diethylstilbesterol, an estrogen agonist, caused only weak inhibition of nitric oxide production, and the effects of resveratrol were not noticeably blocked by ICI-182780, an estrogen antagonist. Structure-activity analysis of resveratrol and nine hydroxystilbenes suggests that the structural balance between oxygen functional groups on the benzene rings is important for their activity. Our results suggest that resveratrol might act on other cellular targets as well as NF-kappaB at the initial stage of gene expression. Unique structural features of hydroxystilbenes are needed for suppression of nitric oxide production and it is unlikely that estrogen receptor is involved in it.

P2X7 Receptor-mediated Membrane Blebbing in Salivary Epithelial Cells.

High concentrations of ATP induce membrane blebbing. However, the underlying mechanism involved in epithelial cells remains unclear. In this study, we investigated the role of the P2X7 receptor (P2X7R) in membrane blebbing using Par C5 cells. We stimulated the cells with 5 mM of ATP for 1~2 hrs and found the characteristics of membrane blebbing, a hallmark of apoptotic cell death. In addition, 500 microM Bz-ATP, a specific P2X7R agonist, induced membrane blebbing. However, 300 microM of Ox-ATP, a P2X7R antagonist, inhibited ATP-induced membrane blebbing, suggesting that ATP-induced membrane blebbing is mediated by P2X7R. We found that ATP-induced membrane blebbing was mediated by ROCK I activation and MLC phosphorylation, but not by caspase-3. Five mM of ATP evoked a biphasic [Ca(2+)](i) response; a transient [Ca(2+)](i) peak and sustained [Ca(2+)](i) increase secondary to ATP-stimulated Ca(2+) influx. These results suggest that P2X7R plays a role in membrane blebbing of the salivary gland epithelial cells.

Molecular cloning and functional expression of a sodium bicarbonate cotransporter from guinea-pig parotid glands.

We recently found that the concentration of HCO3- in guinea-pig saliva is very similar to that of human saliva; however, the entity that regulates HCO3- transport has not yet been fully characterized. In order to investigate the mechanism of HCO3- transport, we identified, cloned, and characterized a sodium bicarbonate (Na(+)/HCO3- cotransporter found in guinea-pig parotid glands (gpNBC1). The gpNBC1 gene encodes a 1079-amino acid protein that has 95% and 96% homology with human and mouse parotid NBC1, respectively. Oocytes expressing gpNBC1 were exposed to HCO3- or Na(+)-free solutions, which resulted in a marked change in membrane potentials (V(m)), suggesting that gpNBC1 is electrogenic. Likewise, a gpNBC1-mediated pH recovery was observed in gpNBC1 transfected human hepatoma cells; however, in the presence of 4, 4-diisothiocyanostilbene-2,2-disulfonic acid, a specific NBC1 inhibitor, such changes in V(m) and pH(i) were not observed. Together, the data show that the cloned guinea-pig gene is a functional, as well as sequence homologue of human NBC1.

Expression of the Na+-HCO3- cotransporter and its role in pHi regulation in guinea pig salivary glands.

Patterns of salivary HCO(3)(-) secretion vary and depend on species and gland types. However, the identities of the transporters involved in HCO(3)(-) transport and the underlying mechanism of intracellular pH (pH(i)) regulation in salivary glands still remain unclear. In this study, we examined the expression of the Na(+)-HCO(3)(-) cotransporter (NBC) and its role in pH(i) regulation in guinea pig salivary glands, which can serve as an experimental model to study HCO(3)(-) transport in human salivary glands. RT-PCR, immunohistochemistry, and pH(i) measurements from BCECF-AM-loaded cells were performed. The amiloride-sensitive Na(+)/H(+) exchanger (NHE) played a putative role in pH(i) regulation in salivary acinar cells and also appeared to be involved in regulation in salivary ducts. In addition to NHE, NBC also played a role in pH(i) regulation in both acini and ducts. In the parotid gland, NBC1 was functionally expressed in the basolateral membrane (BLM) of acinar cells and the luminal membrane (LM) of ducts. In the submandibular gland, NBC1 was expressed only in the BLM of ducts. NBC1 expressed in these two types of salivary glands takes up HCO(3)(-) and is involved in pH(i) regulation. Although NBC3 immunoreactivity was also detected in submandibular gland acinar cells and in the ducts of both glands, it is unlikely that NBC3 plays any role in pH(i) regulation. We conclude that NBC1 is functionally expressed and plays a role in pH(i) regulation in guinea pig salivary glands but that its localization and role are different depending on the type of salivary glands.