Transient Activation and Delayed Inhibition of Na+,K+,Cl– Cotransport in ATP-treated C11-MDCK Cells Involve Distinct P2Y Receptor Subtypes and Signaling Mechanisms*

In C11-MDCK cells, which resemble intercalated cells from collecting ducts of the canine kidney, P2Y agonists promote transient activation of the Na+,K+,Cl– cotransporter (NKCC), followed by its sustained inhibition. We designed this study to identify P2Y receptor subtypes involved in dual regulation of this carrier. Real time polymerase chain reaction analysis demonstrated that C11-MDCK cells express abundant P2Y1 and P2Y2 mRNA compared with that of other P2Y receptor subtypes. The rank order of potency of agents (ATP ∼ UTP ≫ 2-(methylthio)-ATP (2MeSATP); adenosine 5′-[β-thio]diphosphate (ADPβS) inactive) indicated that P2Y2 rather than P2Y1 receptors mediate a 3–4-fold activation of NKCC within the first 5–10 min of nucleotide addition. NKCC activation in ATP-treated cells was abolished by the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, calmodulin (CaM) antagonists trifluoroperazine and W-7, and KN-62, an inhibitor of Ca2+/CaM-dependent protein kinase II. By contrast with the transient activation, 30-min incubation with nucleotides produced up to 4–5-fold inhibition of NKCC, and this inhibition exhibited a rank order of potency (2MeSATP > ADPβS > ATP ≫ UTP) typical of P2Y1 receptors. Unlike the early response, delayed inhibition of NKCC occurred in 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-loaded cells and was completely abolished by the P2Y1 antagonists MRS2179 and MRS2500. Transient activation and delayed inhibition of NKCC in C11 cell monolayers were observed after the addition of ATP to mucosal and serosal solutions, respectively. NKCC inhibition triggered by basolateral application of ADPβS was abolished by MRS2500. Our results thus show that transient activation and delayed inhibition of NKCC in ATP-treated C11-MDCK cells is mediated by Ca2+/CaM-dependent protein kinase II- and Ca2+-independent signaling triggered by apical P2Y2 and basolateral P2Y1 receptors, respectively.

[1]  I. Kügelgen.  Pharmacological profiles of cloned mammalian P2Y-receptor subtypes , 2006 .

[2]  E. Delpire,et al.  Characterization of SPAK and OSR1, Regulatory Kinases of the Na-K-2Cl Cotransporter , 2006, Molecular and Cellular Biology.

[3]  E. Delpire,et al.  Volume sensitivity of cation-Cl- cotransporters is modulated by the interaction of two kinases: Ste20-related proline-alanine-rich kinase and WNK4. , 2006, American journal of physiology. Cell physiology.

[4]  P. Insel,et al.  Cl−secretion in ATP‐treated renal epithelial C7–MDCK cells is mediated by activation of P2Y1 receptors, phospholipase A2 and protein kinase A , 2005, The Journal of physiology.

[5]  S. C. Wolff,et al.  The Apical Targeting Signal of the P2Y2 Receptor Is Located in Its First Extracellular Loop* , 2005, Journal of Biological Chemistry.

[6]  G. Gamba.  Molecular physiology and pathophysiology of electroneutral cation-chloride cotransporters. , 2005, Physiological reviews.

[7]  T. K. Harden,et al.  Polarized expression of human P2Y receptors in epithelial cells from kidney, lung, and colon. , 2005, American journal of physiology. Cell physiology.

[8]  K. Jacobson,et al.  Antiaggregatory activity in human platelets of potent antagonists of the P2Y 1 receptor. , 2004, Biochemical pharmacology.

[9]  A. Kilin,et al.  Cell-volume-dependent vascular smooth muscle contraction: role of Na+, K+, 2Cl− cotransport, intracellular Cl− and L-type Ca2+ channels , 2004, Pflügers Archiv.

[10]  V. Taglietti,et al.  The Duration and Amplitude of the Plateau Phase of ATP- and ADP-Evoked Ca2+ Signals Are Modulated by Ectonucleotidases in in situ Endothelial Cells of Rat Aorta , 2004, Journal of Vascular Research.

[11]  T. Hexum,et al.  Effect of the ecto-ATPase inhibitor, ARL 67156, on the bovine chromaffin cell response to ATP. , 2004, European journal of pharmacology.

[12]  S. Silbernagl,et al.  Characterization of two MDCK-cell subtypes as a model system to study principal cell and intercalated cell properties , 1994, Pflügers Archiv.

[13]  G. Burnstock,et al.  Cellular distribution and functions of P2 receptor subtypes in different systems. , 2004, International review of cytology.

[14]  P. Insel,et al.  Purinergic-induced signaling in C11-MDCK cells inhibits the secretory Na-K-Cl cotransporter. , 2003, American journal of physiology. Cell physiology.

[15]  E. Schwiebert,et al.  Extracellular ATP as a signaling molecule for epithelial cells. , 2003, Biochimica et biophysica acta.

[16]  G. Dubyak,et al.  Colocalization of ATP Release Sites and Ecto-ATPase Activity at the Extracellular Surface of Human Astrocytes* , 2003, Journal of Biological Chemistry.

[17]  E. Delpire,et al.  Cation Chloride Cotransporters Interact with the Stress-related Kinases Ste20-related Proline-Alanine-rich Kinase (SPAK) and Oxidative Stress Response 1 (OSR1)* , 2002, The Journal of Biological Chemistry.

[18]  S. Orlov,et al.  c‐Fos Expression in Ouabain‐Treated Vascular Smooth Muscle Cells from rat Aorta: Evidence for an Intracellular‐Sodium‐Mediated, Calcium‐Independent Mechanism , 2002, The Journal of physiology.

[19]  S. Orlov,et al.  Purinergic-Induced Ion Current in Monolayers of C7-MDCK Cells: Role of Basolateral and Apical Ion Transporters , 2002, The Journal of Membrane Biology.

[20]  S. Orlov,et al.  Swelling-Induced K+ Fluxes in Vascular Smooth Muscle Cells are Mediated by Charybdotoxin-Sensitive K+ Channels , 2001, Cellular Physiology and Biochemistry.

[21]  W. C. O'Neill,et al.  Contractile regulation of the Na(+)-K(+)-2Cl(-) cotransporter in vascular smooth muscle. , 2001, American journal of physiology. Cell physiology.

[22]  G. Vassort.  Adenosine 5'-triphosphate: a P2-purinergic agonist in the myocardium. , 2001, Physiological reviews.

[23]  G. Burnstock,et al.  Axial distribution and characterization of basolateral P2Y receptors along the rat renal tubule. , 2000, Kidney international.

[24]  P. Seth,et al.  p27Kip1 is an inducer of intestinal epithelial cell differentiation , 2000 .

[25]  R. Unwin,et al.  P2 receptors in the kidney. , 2000, Journal of the autonomic nervous system.

[26]  M. Williams,et al.  Purinergic and pyrimidinergic receptors as potential drug targets. , 2000, Biochemical pharmacology.

[27]  P. Insel,et al.  Cellular Release of and Response to ATP as Key Determinants of the Set-Point of Signal Transduction Pathways* , 2000, The Journal of Biological Chemistry.

[28]  M. Haas,et al.  The Na-K-Cl cotransporter of secretory epithelia. , 2000, Annual review of physiology.

[29]  S. Orlov,et al.  Purinergic Modulation of Na+,K+,Cl− Cotransport and MAP Kinases is Limited to C11-MDCK Cells Resembling Intercalated Cells from Collecting Ducts , 1999, The Journal of Membrane Biology.

[30]  S. Orlov,et al.  ATP-Induced Inhibition of Na+, K+, Cl− Cotransport in Madin-Darby Canine Kidney Cells: Lack of Involvement of Known Purinoceptor-Coupled Signaling Pathways , 1999, The Journal of Membrane Biology.

[31]  S. Kunapuli,et al.  P2 receptor subtypes in the cardiovascular system. , 1998, The Biochemical journal.

[32]  E. Delpire,et al.  The electroneutral cation-chloride cotransporters. , 1998, The Journal of experimental biology.

[33]  G. Burnstock,et al.  Potential Functional Roles of Extracellular ATP in Kidney and Urinary Tract , 1998, Nephron Experimental Nephrology.

[34]  H. Endou,et al.  Establishment of a mouse clonal early proximal tubule cell line and outer medullary collecting duct cells expressing P2 purinoceptors , 1998, Biochemistry and molecular biology international.

[35]  S. Orlov,et al.  Complete inhibition of Na+, K+, Cl- cotransport in Madin-Darby canine kidney cells by PMA-sensitive protein kinase. , 1998, Biochimica et biophysica acta.

[36]  S. Orlov,et al.  Bumetanide-sensitive Ion Fluxes in Vascular Smooth Muscle Cells: Lack of Functional Na+, K+, 2 Cl− Cotransport , 1996, The Journal of Membrane Biology.

[37]  S. Orlov,et al.  Cell volume in vascular smooth muscle is regulated by bumetanide-sensitive ion transport. , 1996, The American journal of physiology.

[38]  C. Delles,et al.  A highly calcium‐selective cation current activated by intracellular calcium release in MDCK cells. , 1995, The Journal of physiology.

[39]  E. M. Jones,et al.  Cloning of rat and mouse P2Y purinoceptors. , 1995, Biochemical and biophysical research communications.

[40]  W. Rice,et al.  Cloning and expression of the alveolar type II cell P2u-purinergic receptor. , 1995, American journal of respiratory cell and molecular biology.

[41]  S. Gullans,et al.  Molecular cloning and chromosome localization of a putative basolateral Na(+)-K(+)-2Cl- cotransporter from mouse inner medullary collecting duct (mIMCD-3) cells. , 1994, The Journal of biological chemistry.

[42]  G Burnstock,et al.  Nomenclature and Classification of Purinoceptors* , 2005 .

[43]  B. Nilius.  A role for potassium channels in cell proliferation , 1994 .

[44]  M. Breyer,et al.  Hormonal signaling and regulation of salt and water transport in the collecting duct. , 1994, Annual review of physiology.

[45]  H. Schulman,et al.  Neuronal Ca2+/calmodulin-dependent protein kinases. , 1992, Annual review of biochemistry.

[46]  F. Lang,et al.  Cellular mechanisms of ATP‐induced hyperpolarization in renal epitheloid MDCK‐cells , 1991, Journal of cellular physiology.