A role of sodium bicarbonate cotransporter(NBC) in HCO3- formation in human salivary gland acinar cells

The sodium bicarbonate cotransporter (NBC) protein is functionally expressed in salivary glands. In this experiment, we examined the role of NBC in HCO3- formation in human parotid gland acinar cells. Intracellular pH (pHi) was measured in 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) -loaded cells. Acetazolamide (0.1 mM) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 0.5 mM) were used as specific inhibitors of carbonic anhydrase and NBC, respectively. The degree of inhibition was assessed by measuring the pHi recovery rate (△pHi/min) after cell acidification using an ammonium prepulse technique. In control experiments, △pHi/min was 1.40 ± 0.06. Treatment of cells with 0.5 mM DIDS or 0.1 mM acetazolamide significantly reduced △pH/min to 1.14 ± 0.14 and 0.74 ± 0.15, respectively. Simultaneous application of DIDS and acetazolamide further reduced △ pHi/min to 0.47 ± 0.10. Therefore, DIDS and acetazolamide reduced △pHi/min by 19 % and 47 %, respectively, while simultaneous application of both DIDS and acetazolamide caused a reduction in △pHi/min of 67 %. These results suggest that in addition to carbonic anhydrase, NBC also partially contributes to HCO3- formation in human parotid gland acinar cells.This work was supported by the Korea Science & Engineering Foundation (KOSEF) grant funded by Korea government (R13-2008-008-01001-0) through the Oromaxillofacial Dysfunction Research Center for the Elderly(ODRCE) at Seoul National University

Effects of candesartan and propranolol combination therapy versus propranolol monotherapy in reducing portal hypertension

Background/AimsAngiotensin receptor blockers (ARBs) inhibit activated hepatic stellate cell contraction and are thought to reduce the dynamic portion of intrahepatic resistance. This study compared the effects of combined treatment using the ARB candesartan and propranolol versus propranolol monotherapy on portal pressure in patients with cirrhosis in a prospective, randomized controlled trial.MethodsBetween January 2008 and July 2009, 53 cirrhotic patients with clinically significant portal hypertension were randomized to receive either candesartan and propranolol combination therapy (26 patients) or propranolol monotherapy (27 patients). Before and 3 months after the administration of the planned medication, the hepatic venous pressure gradient (HVPG) was assessed in both groups. The dose of propranolol was subsequently increased from 20 mg bid until the target heart rate was reached, and the candesartan dose was fixed at 8 mg qd. The primary endpoint was the HVPG response rate; patients with an HVPG reduction of >20% of the baseline value or to <12 mmHg were defined as responders.ResultsThe mean portal pressure declined significantly in both groups, from 16 mmHg (range, 12-28 mmHg) to 13.5 mmHg (range, 6-20 mmHg) in the combination group (P<0.05), and from 17 mmHg (range, 12-27 mmHg) to 14 mmHg (range, 7-25 mmHg) in the propranolol monotherapy group (P<0.05). However, the medication-induced pressure reduction did not differ significantly between the two groups [3.5 mmHg (range, -3-11 mmHg) vs. 3 mmHg (range, -8-10 mmHg), P=0.674]. The response rate (55.6% vs. 61.5%, P=0.435) and the reductions in mean blood pressure or heart rate also did not differ significantly between the combination and monotherapy groups.ConclusionsThe addition of candesartan (an ARB) to propranolol confers no benefit relative to classical propranolol monotherapy for the treatment of portal hypertension, and is thus not recommended

Glucocorticoids modulate NF-κB-dependent gene expression by up-regulating FKBP51 expression in Newcastle disease virus-infected chickens

FK506-binding protein 51(FKBP51, coded by FKBP5) is a co-chaperone molecule that interacts with the chaperone HSP90 and the glucocorticoid receptor (GR) in an inactive GR complex. It is a negative regulator of glucocorticoid action and is replaced by the positive regulator, FK506-binding protein 52 (FKBP52, coded by FKBP4) when hormone binds to GR, which renders the GR complex active. In this study, we found that the expression of FKBP51 mRNA in 12 organs of Newcastle disease virus (NDV)-infected chickens was robustly induced. The level of corticosterone in NDV-infected chickens was also elevated, approximately 2- to 6.5-fold in the organs compared to non-infected control chickens. The induction of FKBP51 mRNA expression was reproduced by dexamethasone treatment, indicating a role for glucocorticoids in the systemic induction of FKBP51 mRNA expression. In chicken UMNSAH/DF-1 cells, nuclear factor kappaB (NF-κB) was activated in an FKBP51-dependent manner. Regulation of the three NF-κB-dependent, anti-apoptotic genes, bcl-2, bcl-x and bfl-1/A1 was investigated in UMNSAH/DF-1 cells. Dexamethasone treatment of UMNSAH/DF-1 cells resulted in up-regulation of bcl-2, and down-regulation of bcl-x and bfl-1/A1. Expression of FKBP51 also resulted in down-regulation of bfl-1/A1, but had no effect on bcl-2 and bcl-x, suggesting the involvement of glucocorticoid-FKBP51-NF-κB signaling in the regulation of expression of bfl-1/A1 in UMNSAH/DF-1 cells. We observed organ-specific up- or down-regulation of expression of, bcl-2, bcl-x and bfl-1/A1 in NDV-infected and dexamethasone-treated chickens. Differential regulation of bfl-1/A1, bcl-2 and bcl-x upon NDV-infection and dexamethasone treatment suggests that additional factors are involved in the regulation of these genes. These results suggest that systemic elevation of FKBP51 in NDV-infected chickens activates NF-κB, which cooperates with other factors to regulate the expression of NF-κB-dependent genes.The authors thank Dr. Sang Jin Lee, National Livestock Research Institute, for providing Korean native chicken eggs. This work was supported by a grant (20070401-034-027) from BioGreen 21 Program, Rural Development Administration, Republic of Korea

Molecular Cloning and Functional Expression of a Sodium Bicarbonate Cotransporter from Guinea-Pig Parotid Glands

We recently found that the concentration of in guinea-pig saliva is very similar to that of human saliva; however, the entity that regulates transport has not yet been fully characterized. In order to investigate the mechanism of transport, we identified, cloned, and characterized a sodium bicarbonate 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 or Na+-free solutions, which resulted in a marked change in membrane potentials (Vm), 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 Vm and pHi were not observed. Together, the data show that the cloned guinea-pig gene is a functional, as well as sequence homologue of human NBC1.This work was supported by a Korea Research Foundation Grant, KRF-2003-041-E20215

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

Patterns of salivary HCO3– secretion vary and depend on species and gland types. However, the identities of the transporters involved in HCO3– transport and the underlying mechanism of intracellular pH (pHi) regulation in salivary glands still remain unclear. In this study, we examined the expression of the Na-HCO3– cotransporter (NBC) and its role in pHi regulation in guinea pig salivary glands, which can serve as an experimental model to study HCO3– transport in human salivary glands. RT-PCR, immunohistochemistry, and pHi measurements from BCECF-AM-loaded cells were performed. The amiloride-sensitive Na/H exchanger (NHE) played a putative role in pHi 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 pHi 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 HCO3– and is involved in pHi 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 pHi regulation. We conclude that NBC1 is functionally expressed and plays a role in pHi regulation in guinea pig salivary glands but that its localization and role are different depending on the type of salivary glands

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

Patterns of salivary HCO3- secretion vary and depend on species and gland types. However, the identities of the transporters involved in HCO3- transport and the underlying mechanism of intracellular pH (pHi) regulation in salivary glands still remain unclear. In this study, we examined the expression of the Na+- HCO3- cotransporter (NBC) and its role in pHi regulation in guinea pig salivary glands, which can serve as an experimental model to study HCO3- transport in human salivary glands. RT-PCR, immunohistochemistry, and pHi 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 pHi 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 HCO3- 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 pHi regulation. We conclude that NBC1 is functionally expressed and plays a role in pHi regulation in guinea pig salivary glands but that its localization and role are different depending on the type of salivary glands

Ca2+-activated K+ currents of pancreatic duct cells in guinea-pig

There are numerous studies on transepithelial transports in duct cells including Cl- and/or HCO3-. However, studies on transepithelial K+ transport of normal duct cells in exocrine glands are scarce. In the present study, we examined the characteristics of K+ currents in single duct cells isolated from guinea pig pancreas, using a whole-cell patch clamp technique. Both Cl- and K+ conductance were found with KCl rich pipette solutions. When the bath solution was changed to low Cl-, reversal potentials shifted to the negative side, -75±4 mV, suggesting that this current is dominantly selective to K+. We then characterized this outward rectifying K+ current and examined its Ca22+ dependency. The K+ currents were activated by intracellular Ca22+. 100 nM or 500 nM Ca22+ in pipette significantly (P<0.05) increased outward currents (currents were normalized, 76.8?7.9 pA, n=4 or 107.9?35.5 pA, n=6) at +100 mV membrane potential, compared to those with 0 nM Ca2+ in pipette (27.8?3.7 pA, n=6). We next examined whether this K+ current, recorded with 100 nM Ca2+ in pipette, was inhibited by various inhibitors, including Ba2+, TEA and iberiotoxin. The currents were inhibited by 40.4±% (n=3), 87.0±% (n=5) and 82.5±% (n=9) by 1 mM Ba2+, 5 mM TEA and 100 nM iberiotoxin, respectively. Particularly, an almost complete inhibition of the current by 100 nM iberiotoxin further confirmed that this current was activated by intracellular Ca2+. The K+ current may play a role in secretory process, since recycling of K+ is critical for the initiation and sustaining of Cl- or HCO32+ secretion in these cells.This work was supported by a grant of the Korea Health 21 R&D project, Ministry of Health & Welfare, Republic of Korea (Grant No 01-PJ5-PG1-01CH12-0002)

Simple synthesis of functionalized superparamagnetic magnetite/silica core/shell nanoparticles and their application as magnetically separable high-performance biocatalysts

Uniformly sized silica-coated magnetic nanoparticles (magnetite@silica) are synthesized in a simple one-pot process using reverse micelles as nanoreactors. The core diameter of the magnetic nanoparticles is easily controlled by adjusting the w value ([polar solvent]/[surfactant]) in the reverse-micelle solution, and the thickness of the silica shell is easily controlled by varying the amount of tetraethyl orthosilicate added after the synthesis of the magnetite cores. Several grams of monodisperse magnetite@silica nanoparticles can be synthesized without going through any size-selection process. When crosslinked enzyme molecules form clusters on the surfaces of the magnetite@silica nanoparticles, the resulting hybrid composites are magnetically separable, highly active, and stable under harsh shaking conditions for more than 15 days. Conversely, covalently attached enzymes on the surface of the magnetite@silica nanoparticles are deactivated under the same conditions.X11276sciescopu