cAMP-regulated whole cell chloride currents in pancreatic duct cells

1993 ◽  
Vol 264 (3) ◽  
pp. C591-C602 ◽  
Author(s):  
M. A. Gray ◽  
S. Plant ◽  
B. E. Argent
Keyword(s):  
Pancreatic Duct ◽  
Single Cells ◽  
Duct Cell ◽  
Disulfonic Acid ◽  
Apical Plasma Membrane ◽  
Patch Clamp Technique ◽  
Chloride Currents ◽  
Whole Cell ◽  
Duct Cells ◽  
Pancreatic Duct Cells

Using the whole cell configuration of the patch-clamp technique, we have identified an adenosine 3',5'-cyclic monophosphate (cAMP)-regulated chloride conductance in pancreatic duct cells. Basal whole cell currents in single isolated cells were very low (approximately 5 pA/pF) but could be stimulated 17-fold by elevation of intracellular cAMP. The cAMP-activated currents exhibited 1) a high chloride selectivity, 2) a near linear current-voltage relationship, 3) time and voltage independence, 4) block by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and 5) an anion selectivity sequence based on permeability ratios of SCN > NO3 > Br > Cl > I > HCO3 > F > ClO4 > gluconate. Currents in single cells ran down within a few minutes; however, stable chloride currents could be recorded from duct cell clusters in which four or five cells were in electrical communication. We present evidence suggesting that these cAMP-regulated currents are carried by cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. Physiologically, these CFTR channels act in parallel with chloride-bicarbonate exchangers to facilitate bicarbonate secretion across the apical plasma membrane of the duct cell.

Anion selectivity and block of the small-conductance chloride channel on pancreatic duct cells

1990 ◽  
Vol 259 (5) ◽  
pp. C752-C761 ◽  
Author(s):  
M. A. Gray ◽  
C. E. Pollard ◽  
A. Harris ◽  
L. Coleman ◽  
J. R. Greenwell ◽  
...  
Keyword(s):  
Pancreatic Duct ◽  
Plasma Membranes ◽  
Duct Cell ◽  
Disulfonic Acid ◽  
Patch Clamp Technique ◽  
Anion Selectivity ◽  
Duct Cells ◽  
Anion Permeation ◽  
Pancreatic Duct Cells ◽  
Small Conductance

Rat and human pancreatic duct cells have small-conductance Cl- channels in their apical plasma membranes. These channels are regulated by secretin and adenosine 3',5'-cyclic monophosphate and may function in parallel with Cl(-)-HCO3- exchangers to allow HCO3- secretion from the duct cell. Using the patch-clamp technique, we have now determined the anion permeability sequence of the channel as NO3- greater than Br- approximately I- approximately Cl- much greater than HCO3- much greater than gluconate. From this we conclude 1) that anion permeation involves a weak interaction with charged sites inside the channel pore, 2) that because of the low HCO3-/Cl- permeability ratio it is unlikely that significant amounts of HCO3- could be secreted directly via the channel, and 3) that channel permeability may determine the anion selectivity of secretion. We also show that 5-nitro-2-(3-phenylpropylamino)benzoic acid blocks the small-conductance Cl- channel, whereas 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid has no effect.

Transmembrane chloride currents in human atrial myocytes

1996 ◽  
Vol 270 (2) ◽  
pp. C500-C507 ◽  
Author(s):  
G. R. Li ◽  
J. Feng ◽  
Z. Wang ◽  
S. Nattel
Keyword(s):  
Membrane Conductance ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Atrial Myocytes ◽  
Patch Clamp Technique ◽  
Chloride Currents ◽  
Whole Cell ◽  
Human Atrial Myocytes ◽  
Cyclic Monophosphate ◽  
Whole Cell Patch Clamp

The present study was designed to evaluate the presence of basal, swelling-induced, and cAMP-dependent Cl- currents in human atrial myocytes studied with the whole cell patch-clamp technique. Under basal conditions, a small outwardly rectifying background conductance was noted that reversed close to 0 mV and was not altered by Cl- replacement. Isoproterenol (1 microM), forskolin (3 microM), and 8-bromoadenosine 3',5'-cyclic monophosphate (50 microM) did not increase membrane conductance, even when responsiveness to isoproterenol was confirmed by an increase in Ca2+ current and when perforated-patch techniques (nystatin) were used. Exposure to hyposmotic solutions increased cell volume and induced a whole cell conductance that showed outward rectification, was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (100 microM), and responded to changes in Cl- gradient in a fashion consistent with a Cl(-)-selective conductance, with estimated relative permeabilities of 1, 0.25, and 0.07 for Cl-, methanesulfonate, and aspartate, respectively. The results suggest that human atrial cells lack basal and adenosine 3',5'-cyclic monophosphate-dependent Cl- current but manifest a substantial Cl- conductance in the presence of cell swelling.

CFTR and calcium-activated chloride currents in pancreatic duct cells of a transgenic CF mouse

1994 ◽  
Vol 266 (1) ◽  
pp. C213-C221 ◽  
Author(s):  
M. A. Gray ◽  
J. P. Winpenny ◽  
D. J. Porteous ◽  
J. R. Dorin ◽  
B. E. Argent
Keyword(s):  
Cystic Fibrosis ◽  
Pancreatic Duct ◽  
Chloride Channels ◽  
Insertional Mutagenesis ◽  
Secretory Pathway ◽  
Exon Skipping ◽  
Wild Type ◽  
Chloride Currents ◽  
Duct Cells ◽  
Pancreatic Duct Cells

We have studied the cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride currents in pancreatic duct cells isolated from a transgenic cf/cf mouse created by targeted insertional mutagenesis. Adenosine 3',5'-cyclic monophosphate (cAMP)-activated CFTR chloride currents were detected in 78% (29/37) of wild-type cells, in 81% (35/43) of heterozygote cells, and in 61% (29/47) of homozygous cf/cf duct cells (P > 0.05, cf/cf vs. wild-type and heterozygote). The CFTR current density measured at membrane potentials of +/- 60 mV averaged 22-26 pA/pF in wild-type and heterozygote groups but only 13 pA/pF in cells derived from cf/cf animals (P < 0.05, cf/cf vs. wild-type and cf/cf vs. heterozygotes). In contrast, duct cells from animals of all three genotypic groups exhibited calcium-activated chloride currents that were of similar magnitude and up to 11-fold larger than the CFTR currents. We speculate that these transgenic insertional null mice do not develop the pancreatic pathology that occurs in cystic fibrosis patients because their duct cells contain 1) some wild-type CFTR generated by exon skipping and aberrant splicing and 2) a separate anion secretory pathway mediated by calcium-activated chloride channels.

scholarly journals Single cell transcriptome analysis defines heterogeneity of the adult murine pancreatic ductal tree

2020 ◽  
Author(s):  
Audrey M. Hendley ◽  
Arjun A. Rao ◽  
Laura Leonhardt ◽  
Sudipta Ashe ◽  
Jennifer A. Smith ◽  
...  
Keyword(s):  
Bile Duct ◽  
Pancreatic Duct ◽  
Disease Progression ◽  
Duct Cell ◽  
Cell Types ◽  
Lineage Tracing ◽  
Pancreatic Duct Cell ◽  
Duct Cells ◽  
Pancreatic Duct Cells ◽  
Intrapancreatic Bile Duct

ABSTRACTLineage tracing using genetically engineered mouse models is an essential tool for investigating cell-fate decisions of progenitor cells and biology of mature cell types, with relevance to physiology and disease progression. To study disease development, an inventory of an organ’s cell types and understanding of physiologic function is paramount. Here, we performed singlecell RNA sequencing to examine heterogeneity of murine pancreatic duct cells, pancreatobiliary cells, and intrapancreatic bile duct cells. We isolated duct cells within the murine pancreas using a Dolichos biflorus agglutinin (DBA) lectin sorting strategy that labels all pancreatic duct cell types. Our data suggested the substructure of murine pancreatic duct cells is compartmentalized into three subpopulations. We describe an epithelial-mesenchymal transitory axis in our three pancreatic duct subpopulations and identify SPP1 as a regulator of this fate decision as well as human duct cell de-differentiation. Our results further identify functional heterogeneity within pancreatic duct subpopulations by elucidating a role for Geminin in accumulation of DNA damage in the setting of chronic pancreatitis. Our findings implicate diverse functional roles for subpopulations of pancreatic duct cells in maintenance of duct cell identity and disease progression and establish a comprehensive road map of murine pancreatic duct cell, pancreatobiliary cell, and intrapancreatic bile duct cell homeostasis.SIGNIFICANCEMurine models are extensively used for pancreatic lineage tracing experiments and investigation of pancreatic disease progression. Here, we describe the transcriptome of murine pancreatic duct cells, intrapancreatic bile duct cells, and pancreatobiliary cells at single cell resolution. Our analysis defines novel heterogeneity within the pancreatic ductal tree and supports the paradigm that more than one population of pancreatic duct cells harbors progenitor capacity. We identify and validate unique functional properties of subpopulations of pancreatic duct cells including an epithelial-mesenchymal transcriptomic axis and roles in chronic pancreatic inflammation.

Protein kinase C regulates the magnitude and stability of CFTR currents in pancreatic duct cells

1995 ◽  
Vol 268 (4) ◽  
pp. C823-C828 ◽  
Author(s):  
J. P. Winpenny ◽  
H. L. McAlroy ◽  
M. A. Gray ◽  
B. E. Argent
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Pancreatic Duct ◽  
Specific Inhibitor ◽  
Fluid Secretion ◽  
Pancreatic Ducts ◽  
Patch Clamp Technique ◽  
Kinase C ◽  
Duct Cells ◽  
Pancreatic Duct Cells

Activation of protein kinase C (PKC) inhibits adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated fluid secretion in rat pancreatic ducts (N. Ashton, R. L. Evans, and B. E. Argent. J. Physiol. Lond. 452: 99P, 1992). Using the patch-clamp technique, we have investigated whether this inhibition of fluid secretion results from an effect of PKC on cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels. Exposure to 100 nM 4 beta-phorbol 12,13-dibutyrate (PDBu) had no effect on CFTR current density in unstimulated duct cells, but caused a 31% increase in the magnitude of CFTR currents recorded from cells stimulated with cAMP. Furthermore, prolonged (2-4 h) exposure of stimulated duct cells to 100 nM PDBu (a condition that should downregulate PKC) significantly slowed the rate at which CFTR currents run down after establishing a whole cell recording. A similar effect was observed with calphostin C (500 nM), a specific inhibitor of PKC. Thus, although inhibition of ductal fluid secretion by PDBu is unlikely to be explained by an effect on CFTR, modulation of PKC activity can affect both the magnitude and stability of CFTR currents in pancreatic duct cells.

Volume-activated chloride currents in pancreatic duct cells

1995 ◽  
Vol 147 (2) ◽  
Author(s):  
B. Verdon ◽  
J.P. Winpenny ◽  
K.J. Whitfield ◽  
B.E. Argent ◽  
M.A. Gray
Keyword(s):  
Pancreatic Duct ◽  
Chloride Currents ◽  
Duct Cells ◽  
Pancreatic Duct Cells

scholarly journals Single cell transcriptome analysis defines heterogeneity of the murine pancreatic ductal tree

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Audrey Marie Hendley ◽  
Arjun Arkal Rao ◽  
Laura Leonhardt ◽  
Sudipta Ashe ◽  
Jennifer A Smith ◽  
...  
Keyword(s):  
Bile Duct ◽  
Single Cell ◽  
Pancreatic Duct ◽  
Duct Cell ◽  
Cell Types ◽  
Road Map ◽  
Duct Cells ◽  
Pancreatic Duct Cells ◽  
Fate Decision ◽  
Intrapancreatic Bile Duct

To study disease development, an inventory of an organ's cell types and understanding of physiologic function is paramount. Here, we performed single-cell RNA sequencing to examine heterogeneity of murine pancreatic duct cells, pancreatobiliary cells, and intrapancreatic bile duct cells. We describe an epithelial-mesenchymal transitory axis in our three pancreatic duct subpopulations and identify osteopontin as a regulator of this fate decision as well as human duct cell dedifferentiation. Our results further identify functional heterogeneity within pancreatic duct subpopulations by elucidating a role for geminin in accumulation of DNA damage in the setting of chronic pancreatitis. Our findings implicate diverse functional roles for subpopulations of pancreatic duct cells in maintenance of duct cell identity and disease progression and establish a comprehensive road map of murine pancreatic duct cell, pancreatobiliary cell, and intrapancreatic bile duct cell homeostasis.

pH regulatory mechanisms in rat pancreatic ductal cells

1988 ◽  
Vol 254 (6) ◽  
pp. G925-G930 ◽  
Author(s):  
E. L. Stuenkel ◽  
T. E. Machen ◽  
J. A. Williams
Keyword(s):  
Pancreatic Duct ◽  
Exchange Mechanism ◽  
Disulfonic Acid ◽  
Ductal Cells ◽  
Pancreatic Ductal Cells ◽  
Duct Cells ◽  
Pancreatic Duct Cells ◽  
Stilbene Derivatives ◽  
Disulfonic Stilbene ◽  
Intracellular Alkalinization

The mechanisms underlying regulation of intracellular pH (pHi) by rat pancreatic duct cells were studied by use of the pH-sensitive, fluorescent, cytoplasmically trapped dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Cells exhibited a mean pHi of 7.18 +/- 0.14 in bicarbonate-buffered medium, as calculated from the BCECF fluorescence ratio. Removal of extracellular Na (Nao) caused an intracellular acidification that was rapidly reversed by Na replacement and occurred independently of Clo. Amiloride (10(-3) M) reversibly blocked Na-dependent recovery after Na-free-induced acidification. These results demonstrate the presence of a Na+-H+ exchange mechanism in pancreatic duct cells. Replacement of Clo with gluconate caused an intracellular alkalinization that was reversed by replacement of Cl. Application of the disulfonic stilbene derivatives, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and dihydro-DIDS (2 X 10(-4) M), resulted in block of both Cl-dependent recovery from Na-gluconate and the onset of alkalinization of transition from NaCl to Na-gluconate. Chloride-dependent alteration of pHi occurred independently of Nao. These results demonstrate the presence of an anion exchange mechanism consistent with Cl--HCO3- exchange. Thus pancreatic duct cells contain both Na+-H+ and Cl--HCO3- exchangers.

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