Regulation of Intracellular Ca 2+ by CFTR in Chinese Hamster Ovary Cells

Abstract

In cystic fibrosis, the mutation of the CFTR protein causes reduced transepithelial Cl(-) secretion. As recently proposed, beside its role of Cl(-) channel, CFTR may regulate the activity of other channels such as a Ca(2+)-activated Cl(-) channel. Using a calcium imaging system, we show, in adenovirus-CFTR infected Chinese Hamster Ovary (CHO) cell monolayers, that CFTR can act as a regulator of intracellular [Ca(2+)](i) ([Ca(2+)](i)), involving purino-receptors. Apical exposure to ATP or UTP produced an increase in ([Ca(2+)](i) in noninfected CHO cell monolayers (CHO-WT), in CHO monolayers infected with an adenovirus-CFTR (CHO-CFTR) or infected with an adenovirus-LacZ (CHO-LacZ). The transient [Ca(2+)](i) increase produced by ATP or UTP could be mimicked by activation of CFTR with forskolin (20 microm) in CHO-CFTR confluent monolayers. However, forskolin had no significant effect on [Ca(2+)](i) in noninfected CHO-WT or in CHO-LacZ cells. Pretreatment with purino-receptor antagonists such as suramin (100 microm) or reactive blue-2. (100 microm), and with hexokinase (0.28 U/mg) inhibited the [Ca(2+)](i) response to forskolin in CHO-CFTR infected cells. Taken together, our experiments provide evidence for purino-receptor activation by ATP released from the cell and regulation of [Ca(2+)](i) by CFTR in CHO epithelial cell membranes.

Purinergic signalling in the reproductive system in health and disease

“…This ATP receptor is also linked to the mobilisation of [Ca 2+ ] i and through pharmacological characterisation it appears to be a P2Y 2 and/or P2Y 4 receptor [178]. Exposure to ATP or UTP on the apical side of confluent CHO cell monolayers produced an increase in [Ca 2+ ] i [407]. It was also shown that functional expression of human CFTR in the plasma membrane of CHO cells infected with adenovirus regulated the increase in intracellular Ca 2+ produced by ATP released from the cells.…”

Pharmacological and Signaling Properties of Endogenous P2Y1 Receptors in Cystic Fibrosis Transmembrane Conductance Regulator-Expressing Chinese Hamster Ovary Cells

“…However, the pleiotropic effects due to CFTR dysfunctions and observed on epithelial ion transport in CF largely exceed the impairment of the Cl Ϫ channel function and may be related to other regulatory properties of CFTR. Indeed, in addition to functioning as a cAMP-regulated Cl Ϫ channel, CFTR has been shown to regulate several ion channels by notably activating P2YR subsequent to ATP release (Schwiebert et al, 1995;Urbach and Harvey, 1999).…”

“…However, the pleiotropic effects due to CFTR dysfunctions and observed on epithelial ion transport in CF largely exceed the impairment of the Cl Ϫ channel function and may be related to other regulatory properties of CFTR. Indeed, in addition to functioning as a cAMP-regulated Cl Ϫ channel, CFTR has been shown to regulate several ion channels by notably activating P2YR subsequent to ATP release (Schwiebert et al, 1995;Urbach and Harvey, 1999).Extracellular nucleotides are important signaling moleThis work was supported by institutional grants from the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM), and by the French association Vaincre la Mucoviscidose. B.M.…”

Cystic Fibrosis Transmembrane Conductance Regulator Facilitates ATP Release by Stimulating a Separate ATP Release Channel for Autocrine Control of Cell Volume Regulation

“…Purinergic agonists are essential for many specialized physiological functions (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). In cystic fibrosis (CF), 1 ATP and a related triphosphate nucleotide, UTP, stimulate epithelial chloride (Cl Ϫ ) channels alternative to CFTR via purinergic receptors (11)(12)(13)(14)(15)(16).…”