Coupling Modes and Stoichiometry of Cl−/HCO3− Exchange by slc26a3 and slc26a6

The SLC26 transporters are a family of mostly luminal Cl− and HCO3− transporters. The transport mechanism and the Cl−/HCO3− stoichiometry are not known for any member of the family. To address these questions, we simultaneously measured the HCO3− and Cl− fluxes and the current or membrane potential of slc26a3 and slc26a6 expressed in Xenopus laevis oocytes and the current of the transporters expressed in human embryonic kidney 293 cells. slc26a3 mediates a coupled 2Cl−/1HCO3− exchanger. The membrane potential modulated the apparent affinity for extracellular Cl− of Cl−/HCO3− exchange by slc26a3. Interestingly, the replacement of Cl− with NO3− or SCN− uncoupled the transport, with large NO3− and SCN− currents and low HCO3− transport. An apparent uncoupled current was also developed during the incubation of slc26a3-expressing oocytes in HCO3−-buffered Cl−-free media. These findings were used to develop a turnover cycle for Cl− and HCO3− transport by slc26a3. Cl− and HCO3− flux measurements revealed that slc26a6 mediates a 1Cl−/2HCO3− exchange. Accordingly, holding the membrane potential at 40 and −100 mV accelerated and inhibited, respectively, Cl−-mediated HCO3− influx, and holding the membrane potential at −100 mV increased HCO3−-mediated Cl− influx. These findings indicate that slc26a6 functions as a coupled 1Cl−/2HCO3− exchanger. The significance of isoform-specific Cl− and HCO3− transport stoichiometry by slc26a3 and slc26a6 is discussed in the context of diseases of epithelial Cl− absorption and HCO3− secretion

Glucose transport in interlobular ducts isolated from rat pancreas

Pancreatic duct cells express Na+-dependent glucose transporter, SGLT1 and Na+-independent glucose transporters, GLUT1, GLUT2, and GLUT8. We examined transepithelial glucose transport by pancreatic duct. Interlobular ducts were isolated from rat pancreas. During overnight culture both ends of the duct segments sealed spontaneously. The lumen of the sealed duct was micropunctured and the luminal fluid was replaced by HEPES-buffered solution containing 10.0 mM or 44.4 mM glucose. The bath was perfused with HEPES-buffered solution at 37℃ containing 10.0 or 44.4 mM glucose. Transepithelial differences in osmolality were balanced with mannitol. Glucose transport across ductal epithelium was measured by monitoring changes in luminal volume. When the lumen was filled with 44.4 mM glucose, with either 10.0 or 44.4 mM glucose in the bath, the luminal volume decreased to 65～70% of the initial volume in 15 min. Luminally-injected phlorizin, an inhibitor of SGLT1, abolished the decrease in luminal volume. With 10.0 mM glucose in the lumen and 44.4 mM glucose in the bath, the luminal volume did not change significantly. Luminal application of phlorizin under identical condition led to an increase in luminal volume. The data suggest that both active and passive transport mechanisms of glucose are present in pancreatic ductal epithelium

Aquaporin 1 water channel is overexpressed in the plasma membranes of pancreatic ducts in patients with autoimmune pancreatitis

Chronic pancreatitis with all kinds of etiologies is characterized by pancreatic exocrine dysfunction especially impaired fluid secretion from pancreatic ducts. However, the molecular mechanism of this impaired fluid secretion in chronic pancreatitis is largely unknown. Aquaporin water channels are intrinsic membrane proteins expressed most of the cell types which have high osmotic water permeability. Among them aquaporin 1 (AQP1) is a predominant water channel expressed in the plasma membranes of human pancreatic ducts. Exocrine function test revealed that fluid secretion was severely impaired in AIP. immunohistochemical analysis revealed that AQP1 is localized mainly in the apical and lateral membranes of small pancreatic ducts in control subjects. AQP1 expression was significantly increased in plasma membranes of pancreatic ducts in AIP. Upregulation of AQP1 expression seen in pancreatic ducts of patient with AIP may be caused by the reduced fluid secretion from the pancreas as compensation. Further study would be required to elucidate the precise molecular mechanism for the role of AQP1 in pancreatic fluid secretion from the pancreas in diseases characterized by the impaired ductal fluid secretion such as cystic fibrosis

CFTR Functions as a Bicarbonate Channel in Pancreatic Duct Cells

Pancreatic duct epithelium secretes a HCO3−-rich fluid by a mechanism dependent on cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane. However, the exact role of CFTR remains unclear. One possibility is that the HCO3− permeability of CFTR provides a pathway for apical HCO3− efflux during maximal secretion. We have therefore attempted to measure electrodiffusive fluxes of HCO3− induced by changes in membrane potential across the apical membrane of interlobular ducts isolated from the guinea pig pancreas. This was done by recording the changes in intracellular pH (pHi) that occurred in luminally perfused ducts when membrane potential was altered by manipulation of bath K+ concentration. Apical HCO3− fluxes activated by cyclic AMP were independent of Cl− and luminal Na+, and substantially inhibited by the CFTR blocker, CFTRinh-172. Furthermore, comparable HCO3− fluxes observed in ducts isolated from wild-type mice were absent in ducts from cystic fibrosis (ΔF) mice. To estimate the HCO3− permeability of the apical membrane under physiological conditions, guinea pig ducts were luminally perfused with a solution containing 125 mM HCO3− and 24 mM Cl− in the presence of 5% CO2. From the changes in pHi, membrane potential, and buffering capacity, the flux and electrochemical gradient of HCO3− across the apical membrane were determined and used to calculate the HCO3− permeability. Our estimate of ∼0.1 µm sec−1 for the apical HCO3− permeability of guinea pig duct cells under these conditions is close to the value required to account for observed rates of HCO3− secretion. This suggests that CFTR functions as a HCO3− channel in pancreatic duct cells, and that it provides a significant pathway for HCO3− transport across the apical membrane

Microtubule Dynamics Regulate Cyclic Stretch-Induced Cell Alignment in Human Airway Smooth Muscle Cells

Microtubules are structural components of the cytoskeleton that determine cell shape, polarity, and motility in cooperation with the actin filaments. In order to determine the role of microtubules in cell alignment, human airway smooth muscle cells were exposed to cyclic uniaxial stretch. Human airway smooth muscle cells, cultured on type I collagen-coated elastic silicone membranes, were stretched uniaxially (20% in strain, 30 cycles/min) for 2 h. The population of airway smooth muscle cells which were originally oriented randomly aligned near perpendicular to the stretch axis in a time-dependent manner. However, when the cells treated with microtubule disruptors, nocodazole and colchicine, were subjected to the same cyclic uniaxial stretch, the cells failed to align. Lack of alignment was also observed for airway smooth muscle cells treated with a microtubule stabilizer, paclitaxel. To understand the intracellular mechanisms involved, we developed a computational model in which microtubule polymerization and attachment to focal adhesions were regulated by the preexisting tensile stress, pre-stress, on actin stress fibers. We demonstrate that microtubules play a central role in cell re-orientation when cells experience cyclic uniaxial stretching. Our findings further suggest that cell alignment and cytoskeletal reorganization in response to cyclic stretch results from the ability of the microtubule-stress fiber assembly to maintain a homeostatic strain on the stress fiber at focal adhesions. The mechanism of stretch-induced alignment we uncovered is likely involved in various airway functions as well as in the pathophysiology of airway remodeling in asthma

Protection of Macaques with Diverse MHC Genotypes against a Heterologous SIV by Vaccination with a Deglycosylated Live-Attenuated SIV

HIV vaccine development has been hampered by issues such as undefined correlates of protection and extensive diversity of HIV. We addressed these issues using a previously established SIV-macaque model in which SIV mutants with deletions of multiple gp120 N-glycans function as potent live attenuated vaccines to induce near-sterile immunity against the parental pathogenic SIVmac239. In this study, we investigated the protective efficacy of these mutants against a highly pathogenic heterologous SIVsmE543-3 delivered intravenously to rhesus macaques with diverse MHC genotypes. All 11 vaccinated macaques contained the acute-phase infection with blood viral loads below the level of detection between 4 and 10 weeks postchallenge (pc), following a transient but marginal peak of viral replication at 2 weeks in only half of the challenged animals. In the chronic phase, seven vaccinees contained viral replication for over 80 weeks pc, while four did not. Neutralizing antibodies against challenge virus were not detected. Although overall levels of SIV specific T cell responses did not correlate with containment of acute and chronic viral replication, a critical role of cellular responses in the containment of viral replication was suggested. Emergence of viruses with altered fitness due to recombination between the vaccine and challenge viruses and increased gp120 glycosylation was linked to the failure to control SIV. These results demonstrate the induction of effective protective immune responses in a significant number of animals against heterologous virus by infection with deglycosylated attenuated SIV mutants in macaques with highly diverse MHC background. These findings suggest that broad HIV cross clade protection is possible, even in hosts with diverse genetic backgrounds. In summary, results of this study indicate that deglycosylated live-attenuated vaccines may provide a platform for the elucidation of correlates of protection needed for a successful HIV vaccine against diverse isolates

Climate reconstruction since the Little Ice Age by modelling Koryto glacier, Kamchatka Peninsula, Russia

Based on the field data at Koryto glacier, Kamchatka Peninsula, Russia, we constructed a one-dimensional numerical glacier model which fits the behaviour of the glacier. The analysis of meteorological data from the nearby station suggests that the recent rapid retreat of the glacier since the mid-20th century is likely to be due to a decrease in winter precipitation. Using the geographical data of the glacier terminus variations from 1711 to 1930, we reconstructed the fluctuation in the equilibriumline altitude by means of the glacier model.With summer temperatures inferred from tree-ring data, the model suggests that the winter precipitation from the mid-19th to the early 20th century was about 10% less than that at present. This trend is close to consistent with ice-core results from the nearby ice cap in the central Kamchatka Peninsula