Increase in intracellular Cl? concentration by cAMP- and Ca2+-dependent stimulation of M1 collecting duct cells

Abstract

In the lungs of cystic fibrosis (CF) patients, mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) lead to defective Cl- secretion and hyperabsorption of electrolytes. This may be a an important cause for the defective mucociliary clearance in CF lungs. Previous studies have suggested that inhibition of ENaC during activation of CFTR or by purinergic stimulation could be related to an increase in the intracellular [Cl-]i. This was examined in the present study using cultured mouse M1 collecting duct cells transfected with the chloride-sensitive enhanced yellow fluorescent protein YFP(V163S). Calibration experiments showed a linear decrease of YFP fluorescence intensity with increasing [Cl-]i (0-100 mM). Activation of CFTR by isobutyl-1-methylxanthine (IBMX, 100 microM) and forskolin (2 microM) increased [Cl-]i by 9.6+/-1.5 mM (n=35). Similarly, ATP (100 microM) increased [Cl-]i transiently by 9.5+/-2.2 mM (n=17). The increase in [Cl-]i was reduced by the Na+/K+/2 Cl- -cortransporter-1 (NKCC1) blocker azosemide (100 microM), the CFTR blocker SP-303 (50 microM), the blocker of Ca2+-activated Cl- channels DIDS (100 microM) or the ENaC blocker amiloride (10 microM). Changes in YFP(V163S) fluorescence were not due to changes in cell volume or intracellular pH. The present data thus demonstrate an increase in [Cl-]i following stimulation with secretagogues, which could participate in the inhibition of ENaC.

TMEM16 Proteins Produce Volume-regulated Chloride Currents That Are Reduced in Mice Lacking TMEM16A

“…TMEM16 Proteins Are Essential for RVD in HEK293 CellsTo further confirm the role of TMEM16 channels for I Cl,swell and RVD we loaded HEK293 cells with the fluorescence dye calcein (28). Hypotonic bath solution induced cell swelling and decreased calcein fluorescence, which was followed by a 98% recovery after 20 min, indicating RVD (Fig.…”

Traditional uses, chemistry and pharmacology of Croton species (Euphorbiaceae)

“…The authors suggest its use for the treatment of fluid loss in acute watery diarrhea. SP-303 is the active agent of Provir, a C. lechleri derived product with antisecretory properties 206,207 and hence useful to control fluid loss and diarrhea. SP-303 has been indicated particularly for patients of AIDS, common victims of diarrhea.…”

Purinergic regulation of the epithelial Na+ channel

“…Evidence has emerged from recent studies that, indeed, intracellular Cl − concentration ([Cl − ] i )p lays an important role in the regulation of the activity of ENaC in epithelia. 71 The effect of [Cl − ] i on ENaC was first described in our studies in isolated mouse mandibular duct cells. 72,73 Subsequent studies reported a similar negative relationship between Cl − transport 74,75 71 Thus the change in [Cl − ] i during P2Y 2 R stimulation, which is associated with activity of the basoalteral Na + /K + /2Cl − co-transporter,may at least in part, play a role in the inhibitory effect of nucleotides on ENaC.…”

“…71 The effect of [Cl − ] i on ENaC was first described in our studies in isolated mouse mandibular duct cells. 72,73 Subsequent studies reported a similar negative relationship between Cl − transport 74,75 71 Thus the change in [Cl − ] i during P2Y 2 R stimulation, which is associated with activity of the basoalteral Na + /K + /2Cl − co-transporter,may at least in part, play a role in the inhibitory effect of nucleotides on ENaC. Since bumetanide inhibits only the effect of apical UTP on the activity of ENaC in FRTcells ( Figure 4B), it is tempting to speculate that intracellular Cl − might be an important component of the PTX-insensitive P2Y 2 Rs ignalling pathway that is exclusive tot he receptor in the apical membrane.…”