ABSTRACT
BACKGROUND & AIMS: Cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels play an important role in HCO(3)(-) secretion by pancreatic duct cells (PDCs). Our aims were to characterize the CFTR conductance of guinea pig PDCs and to establish whether CFTR is regulated by HCO(3)(-). METHODS: PDCs were isolated from small intralobular and interlobular ducts, and their Cl(- )conductance was studied using the whole-cell patch clamp technique. RESULTS: Activation of a typical CFTR conductance by adenosine 3',5'-cyclic monophosphate (cAMP) was observed in 114 of 204 cells (56%). A larger (10-fold), time- and voltage-dependent Cl(-) conductance was activated in 39 of 204 cells (19%). Secretin had a similar effect. Coexpression of both conductances in the same cell was observed, and both conductances had similar anion selectivity and pharmacology. Extracellular HCO(3)(-) caused a dose-dependent inhibition of both currents (K(i), approximately 7 mmol/L), which was independent of intracellular and extracellular pH, and the PCO(2) and CO(3)(2-) content of the bathing solutions. CONCLUSIONS: Two kinetically distinct Cl(-) conductances are activated by cAMP in guinea pig PDCs. Because these conductances are coexpressed and exhibit similar characteristics (anion selectivity, pharmacology, and HCO(3)(-) inhibition), we conclude that CFTR underlies them both. The inhibition of CFTR by HCO(3)(-) has implications for the current model of pancreatic ductal HCO(3)(-) secretion.
Subject(s)
Bicarbonates/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis/metabolism , Pancreatic Ducts/metabolism , Sodium Bicarbonate/pharmacokinetics , Animals , Anions/pharmacokinetics , Carbon Dioxide/metabolism , Chloride Channels/metabolism , Chlorides/metabolism , Colforsin/pharmacology , Cyclic AMP/pharmacology , Electric Conductivity , Electrophysiology , Female , Guinea Pigs , Hydrogen-Ion Concentration , Male , Membrane Potentials/drug effects , Membrane Potentials/physiology , Pancreatic Ducts/chemistry , Pancreatic Juice/metabolism , Secretin/pharmacologyABSTRACT
The regulation of chloride efflux from cystic fibrosis pancreatic adenocarcinoma cells (CFPAC-1) and wild-type CFTR-transfected CFPAC-1 cells (TPAC) was compared. Forskolin (10 microM) stimulated chloride efflux from the corrected TPAC cells but not from CFPAC-1 cells. Chloride efflux from both cell types was activated by thapsigargin (0.5 microM). The nucleotides ATP and UTP and the non-hydrolyzable ATP analogue, adenosine 5'-O-(3-thio) triphosphate (ATPgammaS), stimulated chloride efflux from both cell types. None of the other P2 purinoceptor agonists investigated elicited a response. The order of potency was ATP > or = UTP > or = ATPgammaS. Adenosine (10-100 microM) activated choride efflux from the TPAC but not the CFPAC cell line with no increase in intracellular cyclic AMP. Small but statistically significant inhibitions of the adenosine-(50 microM)-stimulated increase in chloride efflux were elicited by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX, 100 nM) and the A2 receptor antagonist 3,7-dimethyl-1-propylargylxanthine (DMPX, 10 microM). The A2A receptor antagonist 8-(3-chlorostyryl)caffeine (CSC, 100 nM) had no significant effect. These results provide evidence for the regulation of chloride efflux by P2Y2 purinoceptors in genetically-corrected and CF pancreatic cell lines. Studies with adenosine receptor antagonists indicate some possible involvement of A1 and A2 (but not A2A) receptors in the adenosine stimulation of chloride efflux, but the relatively small effects of the inhibitors coupled with lack of increase in cyclic AMP and a response only in the CFTR-transfected cells also suggests a possible direct effect of adenosine on CFTR.
Subject(s)
Chloride Channels/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis/metabolism , Pancreas/metabolism , Purinergic Agonists , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/pharmacology , Cell Line , Chloride Channels/drug effects , Colforsin/pharmacology , Cyclic AMP/metabolism , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Enzyme Inhibitors/pharmacology , Humans , Pancreas/cytology , Receptors, Purinergic/genetics , Thapsigargin/pharmacology , Transfection , Uridine Triphosphate/analogs & derivatives , Uridine Triphosphate/pharmacologyABSTRACT
Sodium transport into human placental brush border membrane vesicles was examined in the presence of an outwardly directed sodium gradient leading to the formation of an intravesicular negative charge. 22Na entered the vesicles in a time dependent fashion. The activation energy of the uptake process was calculated and was found to be 11.2 kcal/mol, similar to the value of ionic diffusion in free solution. Amiloride inhibited Na uptake in a concentration dependent fashion with an IC50 value of 3.08 microM. Neither ouabain nor bumetanide had an effect on Na uptake at concentrations up to 100 or 1000 microM, respectively. The system presented here indicates Na transport via channels without involvement of the Na-K-ATPase or the Na-K-Cl cotransporter. The system may be useful in investigating Na transport defects in cystic fibrosis.
