Stable knockdown of CFTR establishes a role for the channel in P2Y receptor-stimulated anion secretion

Melissa L. Palmer, So Yeong Lee, Dan Carlson, Scott Fahrenkrug, Scott M. O'Grady

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

P2Y receptor regulation of anion secretion was investigated in porcine endometrial gland (PEG) epithelial cells. P2Y2, P2Y4, and P2Y6 receptors were detected in monolayers of PEG cells and immunocytochemistry indicated that P2Y4 receptors were located in the apical membrane. Apical membrane current measurements showed that Ca 2+-dependent and PKC-dependent Cl- channels were activated following treatment with uridine triphosphate (UTP) (5 μM). Current-voltage relationships comparing calcium-dependent and PKC-dependent UTP responses under biionic conditions showed significant differences in selectivity between Cl - and I- for the PKC-dependent conductance (P I/PCl = 0.76), but not for Ca2+-dependent conductance (PI/PCl = 1.02). The I-/Cl - permeability ratio for the PKC-dependent conductance was identical to that measured for 8-cpt cAMP. Furthermore, PKC stimulation using phorbol 12-myristate 13-acetate (PMA) activated an apical membrane Cl- conductance that was blocked by the CFTR selective inhibitor, CFTRinh-172. CFTR silencing, accomplished by stable expression of small hairpin RNAs (shRNA), blocked the PKC-activated conductance associated with UTP stimulation and provided definitive evidence of a role for CFTR in anion secretion. CFTR activation increased the initial magnitude of CT secretion, and provided a more sustained secretory response compared to conditions where only Ca 2+-activated Cl- channels were activated by UTP. Measurements of [cAMP], following UTP and PMA stimulation were not significantly different than untreated controls. Thus, these results demonstrate that UTP and PMA activation of CFTR occurs independently of increases in intracellular cAMP and extend the findings of earlier studies of CFTR regulation by PKC in Xenopus oocytes to a mammalian anion secreting epithelium.

Original languageEnglish (US)
Pages (from-to)759-770
Number of pages12
JournalJournal of cellular physiology
Volume206
Issue number3
DOIs
StatePublished - Mar 1 2006

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Uridine Triphosphate
Anions
Acetates
Membranes
Swine
Purinergic P2Y2 Receptors
Chemical activation
Electric current measurement
Xenopus
Small Interfering RNA
Oocytes
Monolayers
Permeability
Epithelium
Epithelial Cells
Immunohistochemistry
Calcium
Electric potential
phorbol-12-myristate

Cite this

Stable knockdown of CFTR establishes a role for the channel in P2Y receptor-stimulated anion secretion. / Palmer, Melissa L.; Lee, So Yeong; Carlson, Dan; Fahrenkrug, Scott; O'Grady, Scott M.

In: Journal of cellular physiology, Vol. 206, No. 3, 01.03.2006, p. 759-770.

Research output: Contribution to journalArticle

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AB - P2Y receptor regulation of anion secretion was investigated in porcine endometrial gland (PEG) epithelial cells. P2Y2, P2Y4, and P2Y6 receptors were detected in monolayers of PEG cells and immunocytochemistry indicated that P2Y4 receptors were located in the apical membrane. Apical membrane current measurements showed that Ca 2+-dependent and PKC-dependent Cl- channels were activated following treatment with uridine triphosphate (UTP) (5 μM). Current-voltage relationships comparing calcium-dependent and PKC-dependent UTP responses under biionic conditions showed significant differences in selectivity between Cl - and I- for the PKC-dependent conductance (P I/PCl = 0.76), but not for Ca2+-dependent conductance (PI/PCl = 1.02). The I-/Cl - permeability ratio for the PKC-dependent conductance was identical to that measured for 8-cpt cAMP. Furthermore, PKC stimulation using phorbol 12-myristate 13-acetate (PMA) activated an apical membrane Cl- conductance that was blocked by the CFTR selective inhibitor, CFTRinh-172. CFTR silencing, accomplished by stable expression of small hairpin RNAs (shRNA), blocked the PKC-activated conductance associated with UTP stimulation and provided definitive evidence of a role for CFTR in anion secretion. CFTR activation increased the initial magnitude of CT secretion, and provided a more sustained secretory response compared to conditions where only Ca 2+-activated Cl- channels were activated by UTP. Measurements of [cAMP], following UTP and PMA stimulation were not significantly different than untreated controls. Thus, these results demonstrate that UTP and PMA activation of CFTR occurs independently of increases in intracellular cAMP and extend the findings of earlier studies of CFTR regulation by PKC in Xenopus oocytes to a mammalian anion secreting epithelium.

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