P2 purinoceptors regulate calcium-activated chloride and fluid transport in 31EG4 mammary epithelia

Schrader

Journal of Biological Chemistry

et al. 2005

115

The amyloid precursor protein (APP) is proteolytically processed by ␤-and ␥-secretases to release amyloid ␤, the main component in senile plaques found in the brains of patients with Alzheimer disease. Alternatively, APP can be cleaved within the amyloid ␤ domain by ␣-secretase releasing the non-amyloidogenic product sAPP␣, which has been shown to have neuroprotective properties. Several G protein-coupled receptors are known to activate ␣-secretase-dependent processing of APP; however, the role of G protein-coupled nucleotide receptors in APP processing has not been investigated. Here it is demonstrated that activation of the G proteincoupled P2Y 2 receptor (P2Y 2 R) subtype expressed in human 1321N1 astrocytoma cells enhanced the release of sAPP␣ in a time-and dose-dependent manner. P2Y 2 Rmediated sAPP␣ release was dependent on extracellular calcium but was not affected by 1,2-bis(2-aminophenoxy)ethane-N,N,N,-trimethylammonium salt, an intracellular calcium chelator, indicating that P2Y 2 R-stimulated intracellular calcium mobilization was not involved. Inhibition of protein kinase C (PKC) with GF109203 or by PKC down-regulation with phorbol ester pre-treatment had no effect on UTP-stimulated sAPP␣ release, indicating a PKC-independent mechanism. U0126, an inhibitor of the mitogen-activated protein kinase pathway, partially inhibited sAPP␣ release by UTP, whereas inhibitors of Src-dependent epidermal growth factor receptor transactivation by P2Y 2 Rs had no effect. The metalloprotease inhibitors phenanthroline and TAPI-2 and the furin inhibitor decanoyl-ArgVal-Lys-Arg-chloromethylketone also diminished UTPinduced sAPP␣ release. Furthermore, small interfering RNA silencing of an endogenous adamalysin, ADAM10 or ADAM17/TACE, partially suppressed P2Y 2 R-activated sAPP␣ release, whereas treatment of cells with both ADAM10 and ADAM17/TACE small interfering RNAs completely abolished UTP-activated sAPP␣ release. These results may contribute to an understanding of the non-amyloidogenic processing of APP.P2 nucleotide receptors modulate a wide range of physiological responses following their activation by extracellular nucleotides (1, 2). The G protein-coupled P2Y 2 receptor (P2Y 2 R) 1 subtype is fully activated by equivalent concentrations of ATP or UTP (3-5) and is up-regulated in salivary gland models of stress and disease (6 -8) as well as in blood vessels after balloon angioplasty and in collared carotid arteries, where it induces intimal hyperplasia and inflammation by increasing smooth muscle cell proliferation and leukocyte infiltration (9, 10). Moreover, nucleotides are released from damaged cells of all tissues and from excited neurons, aggregating platelets, and contracting smooth muscle under physiological conditions (2, 11).The diversity of cellular responses mediated by P2Y 2 Rs is due in part to unique structural features that enable these receptors to stimulate a variety of signal transduction pathways. In addition to the classical stimulation of G␣ q -dependent phospholipase C (12, 13), the P2Y 2 R c...

Section: Discussionmentioning

confidence: 99%

P2Y2 Nucleotide Receptors Enhance α-Secretase-dependent Amyloid Precursor Protein Processing

Schrader

Journal of Biological Chemistry

et al. 2005

115

American Journal of Physiology-Cell Physiology

“…To date, investigations into fundamental aspects of mammary epithelial physiology have utilized mouse (5,6) and bovine (41) cell lines. Such studies have determined that fluid secretion across the apical membrane of murine mammary epithelial cells could be stimulated by inhibition of the epithelial Na ϩ channel or activation of the cystic fibrosis transmembrane conductance regulator Cl Ϫ channel (5) or Ca 2ϩ -dependent Cl Ϫ channels coupled to P2Y 2 purinoceptors (6).…”

mentioning

confidence: 99%

“…Such studies have determined that fluid secretion across the apical membrane of murine mammary epithelial cells could be stimulated by inhibition of the epithelial Na ϩ channel or activation of the cystic fibrosis transmembrane conductance regulator Cl Ϫ channel (5) or Ca 2ϩ -dependent Cl Ϫ channels coupled to P2Y 2 purinoceptors (6). More recently, the work of Quesnell et al (41) demonstrated that the electrolyte composition at the apical membrane of bovine mammary epithelial cells has profound effects on the distribution of the tight junction protein occludin.…”

mentioning

confidence: 99%

Contribution of KCNQ1 to the regulatory volume decrease in the human mammary epithelial cell line MCF-7

Vantol

Missan²,

Crack³

et al. 2007

Using the human mammary epithelial cell line MCF-7, we have investigated volume-activated changes in response to hyposmotic stress. Switching MCF-7 cells from an isosmotic to a hyposmotic solution resulted in an initial cell swelling response, followed by a regulatory volume decrease (RVD). This RVD response was inhibited by the nonselective K(+) channel inhibitors Ba(2+), quinine, and tetraethylammonium chloride, implicating K(+) channel activity in this volume-regulatory mechanism. Additional studies using chromonol 293B and XE991 as inhibitors of the KCNQ1 K(+) channel, and also a dominant-negative NH(2)-terminal truncated KCNQ1 isoform, showed complete abolition of the RVD response, suggesting that KCNQ1 plays an important role in regulation of cell volume in MCF-7 cells. We additionally confirmed that KCNQ1 mRNA and protein is expressed in MCF-7 cells, and that, when these cells are cultured as a polarized monolayer, KCNQ1 is located exclusively at the apical membrane. Whole cell patch-clamp recordings from MCF-7 cells revealed a small 293B-sensitive current under hyposmotic, but not isosmotic conditions, while recordings from mammalian cells heterologously expressing KCNQ1 alone or KCNQ1 with the accessory subunit KCNE3 reveal a volume-sensitive K(+) current, inhibited by 293B. These data suggest that KCNQ1 may play important physiological roles in the mammary epithelium, regulating cell volume and potentially mediating transepithelial K(+) secretion.

Pflugers Arch - Eur J Physiol

“…A lower concentration of DIDS (100 μM) almost completely blocked the current as well (data not shown). Since previous studies have shown that DIDS can block P2 receptors [4,8,16,34], the elimination of the ATP-and ADP-induced current by DIDS may result from direct blockade of a P2 receptor rather than a Cl − channel. Therefore, another Cl − channel blocker, 9-AC, was used.…”

Section: Extracellular Atp Activates Outwardly Rectifying Currents Omentioning

confidence: 97%

Acidic ATP activates lymphocyte outwardly rectifying chloride channels via a novel pathway

Zhou

Liang

et al. 2004

Using whole-cell patch-clamp techniques we found that ATP activated an outwardly rectifying current in Daudi human B lymphoma cells under acidic conditions. The substitution of Cl- for gluconate(-) shifted the reversal potential, while Cl- channel blockers, 4,4'-diisothiocyanostibene-2,2'-disulfonic acid (DIDS) and 9-anthracene carboxylic acid (9-AC), blocked the current, indicating that ATP induces this current by activating the outwardly rectifying chloride channel (ORCC). The effect of ATP on ORCC was mimicked by ADP, but not by other P2 receptor agonists such as ATPgammaS (a poorly hydrolyzable analog of ATP), 2',3'-O-benzoyl-4-benzoyl-ATP (BzATP), and UTP. The ATP-induced ORCC current was completely blocked by 100 microM suramin (a P2 receptor antagonist), and was partially blocked by 100 microM pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS), which is another P2 receptor antagonist. Neither inactivation of G proteins nor elimination of extracellular Ca2+ affected the ATP-induced current, indicating that G protein-coupled P2Y receptors and Ca(2+)-permeable P2X receptors are not involved. Based on the pharmacological profile and the fact that acidic conditions are required for ATP to activate the ORCC, we suggest that acidic ATP activates the lymphocyte ORCC via a novel pathway, which is not associated with any previously described purinergic receptors.