Sulfur Dioxide Activates Cl-/HCO3 - Exchanger via Sulphenylating AE2 to Reduce Intracellular pH in Vascular Smooth Muscle Cells.

Sulfur dioxide (SO2) is a colorless and irritating gas. Recent studies indicate that SO2 acts as the gas signal molecule and inhibits vascular smooth muscle cell (VSMC) proliferation. Cell proliferation depends on intracellular pH (pHi). Transmembrane cystein mutation of Na+- independent Cl-/HCO3 - exchanger (anion exchanger, AE) affects pHi. However, whether SO2 inhibits VSMC proliferation by reducing pHi is still unknown. Here, we investigated whether SO2 reduced pHi to inhibit the proliferation of VSMCs and explore its molecular mechanisms. Within a range of 50-200 µM, SO2 was found to lower the pHi in VSMCs. Concurrently, NH4Cl pre-perfusion showed that SO2 significantly activated AE, whereas the AE inhibitor 4,4'-diisothiocyanatostilbene- 2,20-disulfonic acid (DIDS) significantly attenuated the effect of SO2 on pHi in VSMCs. While 200 µM SO2 sulphenylated AE2, while dithiothreitol (DTT) blocked the sulphenylation of AE2 and subsequent AE activation by SO2, thereby restoring the pHi in VSMCs. Furthermore, DIDS pretreatment eliminated SO2-induced inhibition of PDGF-BB-stimulated VSMC proliferation. We report for the first time that SO2 inhibits VSMC proliferation in part by direct activation of the AE via posttranslational sulphenylation and induction of intracellular acidification.

Role of Pendrin in the Pathophysiology of Aldosterone-Induced Hypertension.

The recent advances in genetics and molecular biology have resulted in the characterization of key components that critically regulate renal NaCl transport and blood pressure. Pendrin is a Cl-/HCO3- exchanger that is highly expressed in thyroid, inner ear, and kidney. In the kidney, it is selectively present at the apical membrane in non-α intercalated cells of the connecting tubules and cortical collecting duct. Besides its role in acid/base homeostasis, accumulating studies using various genetically modified animals have provided compelling evidence that pendrin regulates extracellular fluid volume and electrolyte balance at the downstream of aldosterone signaling. We have shown that angiotensin II and aldosterone cooperatively control pendrin abundance partly through mammalian target of rapamycin signaling and mineralocorticoid receptor dephosphorylation, which is necessary for the kidney to prevent extracellular fluid loss and electrolyte disturbances under physiologic perturbations. In line with the experimental observations, several clinical data indicated that the impaired pendrin function can cause fluid and electrolyte abnormalities in humans. The purpose of this review is to provide an update on the recent progress regarding the role of pendrin in fluid and electrolyte homeostasis, as well as in the pathophysiology of hypertension associated with mineralocorticoid receptor signaling.

ULK1 Phosphorylates and Regulates Mineralocorticoid Receptor.

Mineralocorticoid receptor (MR) signaling regulates both renal Na-Cl reabsorption and K+ excretion. We previously demonstrated that phosphorylation of S843 in the MR ligand-binding domain in renal intercalated cells is involved in the balance of these activities by regulating ligand binding and signaling. However, the kinase that phosphorylates MRS843 is unknown. Using a high-throughput screen assay of 197 kinases, we found that ULK1 is the principal kinase that is responsible for the phosphorylation of MRS843. The results were confirmed by in vitro kinase assay, mass spectrometry, and siRNA knockdown experiments. Notably, phosphorylation at MRS843 was markedly reduced in ULK1/2 double knockout mouse embryonic fibroblasts. Upstream, we show that ULK1 activity is inhibited by phosphorylation induced by angiotensin II via mTOR in cell culture and in vivo. These findings implicate mTOR and ULK1 as regulators of MR activity in intercalated cells, a pathway that is critical for maintaining electrolyte homeostasis.

Functional and molecular mechanism of intracellular pH regulation in human inducible pluripotent stem cells.

AIM: To establish a functional and molecular model of the intracellular pH (pHi) regulatory mechanism in human induced pluripotent stem cells (hiPSCs). METHODS: hiPSCs (HPS0077) were kindly provided by Dr. Dai from the Tri-Service General Hospital (IRB No. B-106-09). Changes in the pHi were detected either by microspectrofluorimetry or by a multimode reader with a pH-sensitive fluorescent probe, BCECF, and the fluorescent ratio was calibrated by the high K+/nigericin method. NH4Cl and Na-acetate prepulse techniques were used to induce rapid intracellular acidosis and alkalization, respectively. The buffering power (ß) was calculated from the ΔpHi induced by perfusing different concentrations of (NH4)2SO4. Western blot techniques and immunocytochemistry staining were used to detect the protein expression of pHi regulators and pluripotency markers. RESULTS: In this study, our results indicated that (1) the steady-state pHi value was found to be 7.5 ± 0.01 (n = 20) and 7.68 ± 0.01 (n =20) in HEPES and 5% CO2/HCO3 --buffered systems, respectively, which were much greater than that in normal adult cells (7.2); (2) in a CO2/HCO3 --buffered system, the values of total intracellular buffering power (ß) can be described by the following equation: ßtot = 107.79 (pHi)2 - 1522.2 (pHi) + 5396.9 (correlation coefficient R 2 = 0.85), in the estimated pHi range of 7.1-8.0; (3) the Na+/H+ exchanger (NHE) and the Na+/HCO3 - cotransporter (NBC) were found to be functionally activated for acid extrusion for pHi values less than 7.5 and 7.68, respectively; (4) V-ATPase and some other unknown Na+-independent acid extruder(s) could only be functionally detected for pHi values less than 7.1; (5) the Cl-/ OH- exchanger (CHE) and the Cl-/HCO3 - anion exchanger (AE) were found to be responsible for the weakening of intracellular proton loading; (6) besides the CHE and the AE, a Cl--independent acid loading mechanism was functionally identified; and (7) in hiPSCs, a strong positive correlation was observed between the loss of pluripotency and the weakening of the intracellular acid extrusion mechanism, which included a decrease in the steady-state pHi value and diminished the functional activity and protein expression of the NHE and the NBC. CONCLUSION: For the first time, we established a functional and molecular model of a pHi regulatory mechanism and demonstrated its strong positive correlation with hiPSC pluripotency.

Regulation of chloride/bicarbonate exchanger on staurosporine-induced mouse cardiomyocyte apoptosis / 中国病理生理杂志

AIM:To investigate whether the chloride/bicarbonate (Cl-/HCO-3) exchanger is involved in staurosporine (STS) induced mouse cardiomyocyte apoptosis,and further understand the function of Cl-/HCO-3 exchanger in cell apoptosis. METHODS:To induce mouse cardiomyocyte apoptosis and to explore the effect of Cl-/HCO-3 exchanger,STS was used with two methods:⑴ different blocker,the chloride channel as well as exchanger blocker DIDS and the chloride channel blocker NPPB; ⑵ different culture medium with or without HCO-3 component. RESULTS:The cell viability and caspase-3 activity of DIDS and NPPB on STS-induced cardiomyocyte apoptosis were 59.7% and 47.2%,175.0% and 212.0% respectively with significant statistic differences (both P0.05,n=20). The cell viability and caspase-3 activity of STS-induced cardiomyocyte apoptosis in culture medium with or without HCO-3 were 29.8% and 41.6%,553.4% and 424.7%,respectively (both P