Effect of Slc26a6 deletion on apical Cl−/HCO3− exchanger activity and cAMP-stimulated bicarbonate secretion in pancreatic duct

2007 ◽  
Vol 292 (1) ◽  
pp. G447-G455 ◽  
Author(s):  
Hiroshi Ishiguro ◽  
Wan Namkung ◽  
Akiko Yamamoto ◽  
Zhaohui Wang ◽  
Roger T. Worrell ◽  
...  
Keyword(s):  
Pancreatic Duct ◽  
Apical Membrane ◽  
Pancreatic Ducts ◽  
Bicarbonate Secretion ◽  
Wild Type ◽  
Rt Pcr ◽  
Secretory Rate ◽  
Transport Fluid ◽  
Exchange Activity

The role of Slc26a6 (PAT1) on apical Cl−/HCO3− exchange and bicarbonate secretion in pancreatic duct cells was investigated using Slc26a6 null and wild-type (WT) mice. Apical Cl−/HCO3− exchange activity was measured with the pH-sensitive dye BCECF in microperfused interlobular ducts. The HCO3−-influx mode of apical [Cl−]i/[HCO3−]o exchange (where brackets denote concentration and subscripts i and o denote intra- and extracellular, respectively) was dramatically upregulated in Slc26a6 null mice ( P < 0.01 vs. WT), whereas the HCO3−-efflux mode of apical [Cl−]o/[HCO3−]i exchange was decreased in Slc26a6 null mice ( P < 0.05 vs. WT), suggesting the unidirectionality of the Slc26a6-mediated HCO3− transport. Fluid secretory rate in interlobular ducts were comparable in WT and Slc26a6 null mice ( P > 0.05). In addition, when pancreatic juice was collected from whole animal in basal and secretin-stimulated conditions, neither juice volume nor its pH showed differences between WT and Slc26a6 null mice. Semiquantitative RT-PCR demonstrated more than fivefold upregulation in Slc26a3 (DRA) expression in Slc26a6 knockout pancreas. In conclusion, these results point to the role of Slc26a6 in HCO3− efflux at the apical membrane and also suggest the presence of a robust Slc26a3 compensatory upregulation, which can replace the function of Slc26a6 in pancreatic ducts.

scholarly journals Functional coupling of apical Cl−/HCO3− exchange with CFTR in stimulated HCO3− secretion by guinea pig interlobular pancreatic duct

2009 ◽  
Vol 296 (6) ◽  
pp. G1307-G1317 ◽  
Author(s):  
A. K. Stewart ◽  
A. Yamamoto ◽  
M. Nakakuki ◽  
T. Kondo ◽  
S. L. Alper ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Pancreatic Duct ◽  
Apical Membrane ◽  
Functional Coupling ◽  
Rt Pcr ◽  
Functional Interaction ◽  
Rich Solution ◽  
Anion Composition ◽  
Apical Membranes ◽  
Exchange Activity

Pancreatic ductal epithelium produces a HCO3−-rich fluid. HCO3− transport across ductal apical membranes has been proposed to be mediated by both SLC26-mediated Cl−/HCO3− exchange and CFTR-mediated HCO3− conductance, with proportional contributions determined in part by axial changes in gene expression and luminal anion composition. In this study we investigated the characteristics of apical Cl−/HCO3− exchange and its functional interaction with Cftr activity in isolated interlobular ducts of guinea pig pancreas. BCECF-loaded epithelial cells of luminally microperfused ducts were alkalinized by acetate prepulse or by luminal Cl− removal in the presence of HCO3−-CO2. Intracellular pH recovery upon luminal Cl− restoration (nominal Cl−/HCO3− exchange) in cAMP-stimulated ducts was largely inhibited by luminal dihydro-DIDS (H2DIDS), accelerated by luminal CFTR inhibitor inh-172 (CFTRinh-172), and was insensitive to elevated bath K+ concentration. Luminal introduction of CFTRinh-172 into sealed duct lumens containing BCECF-dextran in HCO3−-free, Cl−-rich solution enhanced cAMP-stimulated HCO3− secretion, as calculated from changes in luminal pH and volume. Luminal Cl− removal produced, after a transient small depolarization, sustained cell hyperpolarization of ∼15 mV consistent with electrogenic Cl−/HCO3− exchange. The hyperpolarization was inhibited by H2DIDS and potentiated by CFTRinh-172. Interlobular ducts expressed mRNAs encoding CFTR, Slc26a6, and Slc26a3, as detected by RT-PCR. Thus Cl−-dependent apical HCO3− secretion in pancreatic duct is mediated predominantly by an Slc26a6-like Cl−/HCO3− exchanger and is accelerated by inhibition of CFTR. This study demonstrates functional coupling between Cftr and Slc26a6-like Cl−/HCO3− exchange activity in apical membrane of guinea pig pancreatic interlobular duct.

scholarly journals Deletion of Slc26a6 alters the stoichiometry of apical Cl−/HCO3− exchange in mouse pancreatic duct

2012 ◽  
Vol 303 (8) ◽  
pp. C815-C824 ◽  
Author(s):  
Ying Song ◽  
Akiko Yamamoto ◽  
Martin C. Steward ◽  
Shigeru B. H. Ko ◽  
Andrew K. Stewart ◽  
...  
Keyword(s):  
Pancreatic Duct ◽  
Apical Membrane ◽  
Flux Ratio ◽  
Pancreatic Ducts ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Membrane Hyperpolarization ◽  
Disulphonic Acid ◽  
Cystic Fibrosis Transmembrane

To define the stoichiometry and molecular identity of the Cl−/HCO3− exchanger in the apical membrane of pancreatic duct cells, changes in luminal pH and volume were measured simultaneously in interlobular pancreatic ducts isolated from wild-type and Slc26a6-null mice. Transepithelial fluxes of HCO3− and Cl− were measured in the presence of anion gradients favoring rapid exchange of intracellular HCO3− with luminal Cl− in cAMP-stimulated ducts. The flux ratio of Cl− absorption/HCO3− secretion was ∼0.7 in wild-type ducts and ∼1.4 in Slc26a6−/− ducts where a different Cl−/HCO3− exchanger, most likely SLC26A3, was found to be active. Interactions between Cl−/HCO3− exchange and cystic fibrosis transmembrane conductance regulator (CFTR) in cAMP-stimulated ducts were examined by measuring the recovery of intracellular pH after alkali-loading by acetate prepulse. Hyperpolarization induced by luminal application of CFTRinh-172 enhanced HCO3− efflux across the apical membrane via SLC26A6 in wild-type ducts but significantly reduced HCO3− efflux in Slc26a6−/− ducts. In microperfused wild-type ducts, removal of luminal Cl−, or luminal application of dihydro-4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid to inhibit SLC26A6, caused membrane hyperpolarization, which was abolished in Slc26a6−/− ducts. In conclusion, we have demonstrated that deletion of Slc26a6 alters the apparent stoichiometry of apical Cl−/HCO3− exchange in native pancreatic duct. Our results are consistent with SLC26A6 mediating 1:2 Cl−/HCO3− exchange, and the exchanger upregulated in its absence, most probably SLC26A3, mediating 2:1 exchange.

scholarly journals The Na+/H+ exchanger isoform 3 is required for active paracellular and transcellular Ca2+ transport across murine cecum

2013 ◽  
Vol 305 (4) ◽  
pp. G303-G313 ◽  
Author(s):  
Juraj Rievaj ◽  
Wanling Pan ◽  
Emmanuelle Cordat ◽  
R. Todd Alexander
Keyword(s):  
Water Movement ◽  
Water Flux ◽  
Wild Type Mouse ◽  
Wild Type ◽  
Rt Pcr ◽  
Ussing Chambers ◽  
Voltage Clamps ◽  
Paracellular Absorption ◽  
Mouse Intestine

Intestinal calcium (Ca2+) absorption occurs via paracellular and transcellular pathways. Although the transcellular route has been extensively studied, mechanisms mediating paracellular absorption are largely unexplored. Unlike passive diffusion, secondarily active paracellular Ca2+ uptake occurs against an electrochemical gradient with water flux providing the driving force. Water movement is dictated by concentration differences that are largely determined by Na+ fluxes. Consequently, we hypothesized that Na+ absorption mediates Ca2+ flux. NHE3 is central to intestinal Na+ absorption. NHE3 knockout mice (NHE3−/−) display impaired intestinal Na+, water, and Ca2+ absorption. However, the mechanism mediating this latter abnormality is not clear. To investigate this, we used Ussing chambers to measure net Ca2+ absorption across different segments of wild-type mouse intestine. The cecum was the only segment with net Ca2+ absorption. Quantitative RT-PCR measurements revealed cecal expression of all genes implicated in intestinal Ca2+ absorption, including NHE3. We therefore employed this segment for further studies. Inhibition of NHE3 with 100 μM 5-( N-ethyl- N-isopropyl) amiloride decreased luminal-to-serosal and increased serosal-to-luminal Ca2+ flux. NHE3−/− mice had a >60% decrease in luminal-to-serosal Ca2+ flux. Ussing chambers experiments under altered voltage clamps (−25, 0, +25 mV) showed decreased transcellular and secondarily active paracellular Ca2+ absorption in NHE3−/− mice relative to wild-type animals. Consistent with this, cecal Trpv6 expression was diminished in NHE3−/− mice. Together these results implicate NHE3 in intestinal Ca2+ absorption and support the theory that this is, at least partially, due to the role of NHE3 in Na+ and water absorption.

Mechanism of proximal tubule bicarbonate absorption in NHE3 null mice

1999 ◽  
Vol 277 (2) ◽  
pp. F298-F302 ◽  
Author(s):  
Tong Wang ◽  
Chao-Ling Yang ◽  
Thecla Abbiati ◽  
Patrick J. Schultheis ◽  
Gary E. Shull ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Proximal Tubule ◽  
Apical Membrane ◽  
Significant Inhibition ◽  
Fluid Absorption ◽  
Wild Type ◽  
Significant Component ◽  
Nhe Isoforms

NHE3 is the predominant isoform responsible for apical membrane Na+/H+exchange in the proximal tubule. Deletion of NHE3 by gene targeting results in an NHE3−/−mouse with greatly reduced proximal tubule[Formula: see text] absorption compared with NHE3+/+ animals (P. J. Schultheis, L. L. Clarke, P. Meneton, M. L. Miller, M. Soleimani, L. R. Gawenis, T. M. Riddle, J. J. Duffy, T. Doetschman, T. Wang, G. Giebisch, P. S. Aronson, J. N. Lorenz, and G. E. Shull. Nature Genet. 19: 282–285, 1998). The purpose of the present study was to evaluate the role of other acidification mechanisms in mediating the remaining component of proximal tubule [Formula: see text] reabsorption in NHE3−/− mice. Proximal tubule transport was studied by in situ microperfusion. Net rates of[Formula: see text] ( J HCO3) and fluid absorption ( J v) were reduced by 54 and 63%, respectively, in NHE3 null mice compared with controls. Addition of 100 μM ethylisopropylamiloride (EIPA) to the luminal perfusate caused significant inhibition of J HCO3 and J v in NHE3+/+ mice but failed to inhibit J HCO3 or J v in NHE3−/− mice, indicating lack of activity of NHE2 or other EIPA-sensitive NHE isoforms in the null mice. Addition of 1 μM bafilomycin caused a similar absolute decrement in J HCO3 in wild-type and NHE3 null mice, indicating equivalent rates of[Formula: see text] absorption mediated by H+-ATPase. Addition of 10 μM Sch-28080 did not reduce J HCO3 in either wild-type or NHE3 null mice, indicating lack of detectable H+-K+-ATPase activity in the proximal tubule. We conclude that, in the absence of NHE3, neither NHE2 nor any other EIPA-sensitive NHE isoform contributes to mediating [Formula: see text] reabsorption in the proximal tubule. A significant component of[Formula: see text] reabsorption in the proximal tubule is mediated by bafilomycin-sensitive H+-ATPase, but its activity is not significantly upregulated in NHE3 null mice.

The Critical Role of Peptidyl-Prolyl cis/trans Isomerase, Pin1 in Acute Myeloid Leukemia with C/EBPalpha Mutations.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2213-2213
Author(s):  
J. Pulikkan ◽  
A. Peer Zada ◽  
M. Geletu ◽  
V. Dengler ◽  
Daniel G. Tenen ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Dominant Negative ◽  
Pcr Analysis ◽  
Wild Type ◽  
Rt Pcr ◽  
Promoter Assay ◽  
Type C ◽  
Acute Myeloid

Abstract CCAAT enhancer binding protein alpha (C/EBPα) is a myeloid specific transcription factor that coordinates cellular differentiation and cell cycle arrest. Loss of C/EBPα expression or function in leukemic blasts contributes to a block in myeloid cell differentiation. C/EBPα is mutated in around 9% of acute myeloid leukemia (AML). The mutations reported in C/EBPα are frame shift mutations and point mutations at basic region Leucine zipper. The mutant form of C/EBPα ie C/EBPα-p30 exhibits dominant negative function over the wild type protein. The role of peptidyl-prolyl cis/trans isomerase, Pin1 in tumorogenesis and its overexpression in many cancers led us to investigate its role in acute myeloid leukemia with C/EBPα mutation. Here we show that Pin1 is upregulated in patients with acute myeloid leukemia by affymetrix analysis. By quantitative Real-Time RT-PCR analysis, we show C/EBPα-p30 could induce Pin1 transcription, while the wild type C/EBPα downregulates Pin1 expression. Luciferase promoter assay for the Pin1 promoter shows that wild type C/EBPα is able to block Pin1 promoter activity. Mean while, C/EBPα-p30 couldn’t block Pin1 promotor activity. By silencing Pin1 by RNA Interference as well as with inhibitor against Pin1 (PiB) we could show myeloid differentiation in human CD34+ cord blood cells as well as in Kasumi-6 cells as assessed by FACS analysis with granulocytic markers. We investigated the mechanism underlying the dominant negative action of C/EBPα-p30 over the wild type protein. We report that Pin1 increases the transcriptional activity of the oncogene c-jun. We also show that c-jun blocks the DNA binding and transactivation of C/EBPα protein as assessed by gel shift assay and promoter assay respectively. We have previously shown that c-jun expression is high in AML patients with C/EBPα mutation and c-jun could block C/EBPα function by protein-protein interaction. Quantitative Real-Time RT-PCR analysis shows that inhibition of Pin1 by the inhibitor PiB downregulates c-jun mRNA expression. In conclusion, inhibition of Pin1 leads to granulocytic differentiation. Our results show Pin1 as a novel target in treating AML patients with C/EBPα mutation.

PAX5/TEL Exhibits a Dominant-Negative Role by Inhibiting the Expression of Genes Involved in BCR Signalling and Suppressing the Binding of Wild Type PAX5 to Its Direct Target Gene CD79A (mb-1) in a human Pre-B ALL Cell Line.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3455-3455
Author(s):  
Gabriela B. Iwanski ◽  
Nils Heinrich Thoennissen ◽  
PohYeen Lor ◽  
Norihiko Kawamata ◽  
Daniel Nowak ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Fusion Gene ◽  
Cell Receptor ◽  
Dominant Negative ◽  
Fusion Product ◽  
Wild Type ◽  
Rt Pcr ◽  
Downstream Target

Abstract Abstract 3455 Poster Board III-343 Acute lymphoblastic leukemia (ALL), one of the most common malignancies in childhood, is a heterogeneous disease with individual leukemia subtypes differing in their response to chemotherapy. Recent findings suggest that disruptions of B cell receptor (BCR) signalling pathways may be involved in the development of ALL. The transcription factor PAX5 is essential for the commitment of lymphoid progenitors to the B-lymphocytic lineage. In 30% of childhood B-ALL cases, PAX5 is a frequent target of aberrancies, showing monoallelic loss, point mutations, or chromosomal translocations, whereas the role of these aberrancies is still poorly understood. Using high resolution SNP-chip analysis, we have recently identified several candidate partner genes fused to PAX5 in pediatric ALL, ETV6 (TEL), FOXP1, AUTS2, C20orf112, which bind to PAX5 recognition sequences as strongly as wild-type PAX5 (wt PAX5) suppressing its transcriptional activity in a dominant-negative fashion. In order to study the role of PAX5/TEL in leukemic evolution of B-ALL, we transfected the leukemic BCP cell line Nalm 6, which endogenously expresses PAX5, with a retroviral vector encoding PAX5/TEL and confirmed its expression by Western blotting and RT-PCR. Previously, the fusion gene PAX5/TEL has been cloned into the retroviral vector pMSCV-IRES-GFP (MIGR) from a patient diagnosed with B-cell precursor ALL (BCP) with t(9;12)(q11;p13). This fusion product consists of the 5′-end NH2 terminal region of the PAX5 gene and the almost whole sequence of the TEL gene. PAX5/TEL-MIGR expressing cells were sorted for GFP and analyzed by gene expression profiling on Affymetrix HG-U133 plus 2.0 Array in comparison to cells transfected with vector control (MIGR) and a MIGR vector encoding wt PAX5 (wtPAX5/MIGR). The probes were normalized with the Affymetrix MAS5.0 software. Probes were considered to be differentially expressed with a fold change ≤ 2 or ≥ 2, respectively. We identified a set of about 200 genes that were differentially expressed in the PAX5/TEL expressing cells, most of which were downregulated, compared to the controls. A subset of these genes encodes proteins important for BCR signalling: RAG1, one of two key mediators in the process of V(D)J recombination, VPREB3, which is involved in the early phase of pre-BCR assembly, the Runt domain transcription factor Runx1 (AML1) and FOXP1. The latter two genes are fusion partners of PAX5 in pediatric B-ALL and loss of FOXP1 leads to impaired DH–JH and VH–DJH rearrangement. Additionally, we found BACH2, which plays an important role during B-cell development, as well as protein kinase C-epsilon (PKCe) to be downregulated. PKCe is highly expressed in B cells linking the BCR to the activation of mitogen-activated protein kinases (MAPK). We confirmed the downregulation of the affected genes by RT-PCR. Strikingly, VPREB3 expression showed a significant downregulation of up to 170-fold, and RAG1 up to 90-fold. Loss of the RAG1/2 locus has been found in four precursor B-cell ALL cases, which indicates that defects in this process might contribute to leukemogenesis. We also detected a significant decrease in the expression of wt PAX5 as well as its direct downstream target CD79A (mb-1). CD79A (mb-1) encodes the B cell receptor component Ig-a and its early B cell-specific mb-1 promoter is a target for regulation by early B cell-specific transcription factors like E2A, early B cell factor (EBF), and PAX5. The latter is important for the activation of the mb-1 promoter by recruiting Ets proteins through protein-protein interactions. We investigated the binding efficiency of wt PAX5 to the promoter region of CD79A by chromatin-immunoprecipitation (ChIP). For the ChIP assay, we used a PAX5 antibody detecting the C-terminal region of PAX5 so that the antibody can bind the wt PAX5 but not the fusion product PAX5/TEL of which the C-terminal side is fused to TEL. Binding of wt PAX5 to the promoter region of CD79A was diminished by expression of the PAX5/TEL-fusion protein compared to the controls, leading to repression of CD79A, which we also confirmed by RT-PCR. In conclusion, we show that the expression of PAX5/TEL in a leukemic cell line has a repressor function on the expression of wt PAX5 as well as other genes important in BCR signalling. Also, we demonstrated that PAX5/TEL has a negative impact on the binding affinity of one of the direct downstream target genes of wt PAX5. Our results indicate a repressor role of the fusion gene PAX5/TEL including BCR signalling and point towards its contribution to leukemic transformation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Both NHERF3 and NHERF2 are necessary for multiple aspects of acute regulation of NHE3 by elevated Ca2+, cGMP, and lysophosphatidic acid

2018 ◽  
Vol 314 (1) ◽  
pp. G81-G90 ◽  
Author(s):  
Leela Rani Avula ◽  
Tiane Chen ◽  
Olga Kovbasnjuk ◽  
Mark Donowitz
Keyword(s):  
Brush Border ◽  
Apical Membrane ◽  
Cell Model ◽  
Pdz Domain ◽  
Intestinal Epithelial ◽  
Wild Type ◽  
Dual Emission ◽  
Two Photon Microscopy ◽  
Two Photon

The intestinal epithelial brush border Na+/H+ exchanger NHE3 accounts for a large component of intestinal Na absorption. NHE3 is regulated during digestion by signaling complexes on its COOH terminus that include the four multi-PDZ domain-containing NHERF family proteins. All bind to NHE3 and take part in different aspects of NHE3 regulation. Because the roles of each NHERF appear to vary on the basis of the cell model or intestinal segment studied and because of our recent finding that a NHERF3-NHERF2 heterodimer appears important for NHE3 regulation in Caco-2 cells, we examined the role of NHERF3 and NHERF2 in C57BL/6 mouse jejunum using homozygous NHERF2 and NHERF3 knockout mice. NHE3 activity was determined with two-photon microscopy and the dual-emission pH-sensitive dye SNARF-4F. The jejunal apical membrane of NHERF3-null mice appeared similar to wild-type (WT) mice in surface area, microvillus number, and height, which is similar to results previously reported for jejunum of NHERF2-null mice. NHE3 basal activity was not different from WT in either NHERF2- or NHERF3-null jejunum, while d-glucose-stimulated NHE3 activity was reduced in NHERF2, but similar to WT in NHERF3 KO. LPA stimulation and UTP (elevated Ca2+) and cGMP inhibition of NHE3 were markedly reduced in both NHERF2- and NHERF3-null jejunum. Forskolin inhibited NHE3 in NHERF3-null jejunum, but the extent of inhibition was reduced compared with WT. The forskolin inhibition of NHE3 in NHERF2-null mice was too inconsistent to determine whether there was an effect and whether it was altered compared with the WT response. These results demonstrate similar requirement for NHERF2 and NHERF3 in mouse jejunal NHE3 regulation by LPA, Ca2+, and cGMP. The explanation for the similarity is not known but is consistent with involvement of a brush-border NHERF3-NHERF2 heterodimer or sequential NHERF-dependent effects in these aspects of NHE3 regulation. NEW & NOTEWORTHY NHERF2 and NHERF3 are apical membrane multi-PDZ domain-containing proteins that are involved in regulation of intestinal NHE3. This study demonstrates that NHERF2 and NHERF3 have overlapping roles in NHE3 stimulation by LPA and inhibition by elevated Ca2+ and cGMP. These results are consistent with their role being as a NHERF3-NHERF2 heterodimer or via sequential NHERF-dependent signaling steps, and they begin to clarify a role for multiple NHERF proteins in NHE3 regulation.

PAT-1 (Slc26a6) is the predominant apical membrane Cl−/HCO3− exchanger in the upper villous epithelium of the murine duodenum

2007 ◽  
Vol 292 (4) ◽  
pp. G1079-G1088 ◽  
Author(s):  
Janet E. Simpson ◽  
Clifford W. Schweinfest ◽  
Gary E. Shull ◽  
Lara R. Gawenis ◽  
Nancy M. Walker ◽  
...  
Keyword(s):  
Anion Exchanger ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Duodenal Mucosa ◽  
Transport Processes ◽  
Wild Type ◽  
Rt Pcr ◽  
Targeted Deletion ◽  
Anion Transporter ◽  
Dependent Transport

Basal HCO3− secretion across the duodenum has been shown in several species to principally involve the activity of apical membrane Cl−/HCO3− exchanger(s). To investigate the identity of relevant anion exchanger(s), experiments were performed using wild-type (WT) mice and mice with gene-targeted deletion of the following Cl−/HCO3− exchangers localized to the apical membrane of murine duodenal villi: Slc26a3 [down-regulated in adenoma (DRA)], Slc26a6 [putative anion transporter 1 (PAT-1)], and Slc4a9 [anion exchanger 4 (AE4)]. RT-PCR of the isolated villous epithelium demonstrated PAT-1, DRA, and AE4 mRNA expression. Using the pH-sensitive dye BCECF, anion exchange rates were measured across the apical membrane of epithelial cells in the upper villus of the intact duodenal mucosa. Under basal conditions, Cl−/HCO3− exchange activity was reduced by 65–80% in the PAT-1(−) duodenum, 30–40% in the DRA(−) duodenum, and <5% in the AE4(−) duodenum compared with the WT duodenum. SO42−/HCO3− exchange was eliminated in the PAT-1(−) duodenum but was not affected in the DRA(−) and AE4(−) duodenum relative to the WT duodenum. Intracellular pH (pHi) was reduced in the PAT-1(−) villous epithelium but increased to WT levels in the absence of CO2/HCO3− or during methazolamide treatment. Further experiments under physiological conditions indicated active pHi compensation in the PAT-1(−) villous epithelium by combined activities of Na+/H+ exchanger 1 and Cl−-dependent transport processes at the basolateral membrane. We conclude that 1) PAT-1 is the major contributor to basal Cl−/HCO3− and SO42−/HCO3− exchange across the apical membrane and 2) PAT-1 plays a role in pHi regulation in the upper villous epithelium of the murine duodenum.

The Role of the cGMP-Dependent Protein Kinase II in Platelet Activation and In Vivo Thrombosis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 653-653
Author(s):  
Zhenyu Li ◽  
Guoying Zhang ◽  
Hong Yin ◽  
Robert Feil ◽  
Franz Hofmann ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Low Dose ◽  
Bleeding Time ◽  
Wild Type ◽  
Rt Pcr ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein

Abstract Although it was previously believed that the intracellular secondary messenger cGMP inhibits platelets, we have recently shown that cGMP-dependent protein kinase I (PKG I) in fact plays a stimulatory role in platelet activation. However, there are apparent differences between the PKG inhibitors and PKG I knockout in their effects on platelet activation. PKG inhibitors are more potent in inhibiting platelet activation than PKG I knockout. More importantly, although platelet secretion and aggregation induced by collagen were inhibited by PKG inhibitors, they are not significantly affected in PKG I knockout platelets. There are two types of PKG, PKG I and PKG II. PKG II has not been previously described in platelets. Here we show that PKG II mRNA is expressed in platelets using RT-PCR with primers specific for a C-terminal fragment of human PKG II cDNA. We further cloned the complete cDNA of human PKG II by RT-PCR using the purified human platelet mRNA as a template. Furthermore, PKG II from platelet lysates was pulled down by cGMP conjugated agarose beads and detected by western blot using a polyclonal antibody against PKG II. These data indicate that PKG II is expressed in platelets. To investigate the role of PKG II in platelet activation, washed wild type or PKG II knockout (PKG II−/−) mouse platelets in tyrode’s solution were exposed to platelet agonists. Platelet aggregation and ATP secretion induced by low concentrations of collagen were significantly reduced in PKG II deficient mice, indicating that PKG II plays important roles in collagen-induced platelet activation. PKG II−/− platelets also showed reduced aggregation and secretion to low dose of a thromboxane A2 (TXA2) analog, U46619. However, low dose thrombin-induced platelet activation was not negatively affected in PKG II−/− platelets, but was inhibited in PKG I−/− platelets. To evaluate the in vivo role of PKG II, we compared in vivo thrombus formation of wild type and PKG II knockout mice using the FeCl3-injured carotid artery thrombosis model. The time to the formation of stable thrombus in PKG II−/− mice (median, 420.0 seconds, n=15) is significantly prolonged compared to wild type mice (median, 321.0 seconds, n=15) (p=0.031). Tail-bleed time analysis also indicated a remarkably prolonged bleeding time in PKG II−/− mice (the median bleeding time was 73.50 seconds (n=18) in wild type mice, 454.50 seconds (n=20) in PKG II knockout mice) (p=0.0008). Thus, PKG II plays an important role in promoting platelet activation, thrombosis and hemostasis. PKG I and PKG II have differential roles in platelet activation induced by different platelet agonists.

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