Cl- secretion in ATP-treated renal epithelial C7-MDCK cells is mediated by activation of P 2Y1 receptors, phospholipase A2 and protein kinase A

Abstract

This study examines the mechanism of P 2Y-induced Cl- secretion in monolayers of C7-Madin-Darby canine kidney (MDCK) cells triggered by basolateral application of ATP and measured as transcellular short current (I(SC)). Both ATP-induced arachidonic acid (AA) synthesis and I(SC) in ATP-treated cells were abolished by the phosholipase A2 (PLA2) inhibitor, AACOCF3. The cyclo-oxygenase inhibitor indomethacin decreased I(SC) and cAMP production in ATP-treated cells with an IC50 of approximately 0.3 microm. ATP led to rapid activation of cAMP-dependent protein kinase A (PKA), as estimated by phosphorylation of a vasodilator-stimulated phosphoprotein. PKA activity and I(SC) evoked by ATP, as well as by prostaglandin E1 (PGE1), were diminished in the presence of the PKA inhibitor H-89 or an adenovirus-mediated expression of PKA-inhibitor protein, PKI. In contrast, indomethacin completely blocked the increment of PKA and I(SC) triggered by ATP and AA, but did not affect PKA activation and I(SC) detected with PGE1. The kinetics of [Ca2+]i elevation in ATP- and thapsigargin-treated cells were similar and suppressed by the Ca(2+)i chelator BAPTA. Neither baseline nor maximal increment of ATP-induced I(SC) was affected by thapsigargin and BAPTA. Real-time PCR showed that C7 cells express more mRNA for P 2Y1 and P 2Y2 than for other P 2Y receptor subtypes. The rank order of potency (2MeSATP > ATP > ADP >> UTP) indicates that P 2Y1 rather than P 2Y2 receptors contribute to PKA and I(SC) activation. Viewed collectively, these data show that Cl- secretion in C7-MDCK monolayers treated with basolateral ATP is triggered by P 2Y1 receptors and is mediated by subsequent [Ca2+]i-independent activation of PLA2 and PKA.

Figure 1. Short-circuit current in monolayers of c7-MDCK cell

Typical records of I_SC modulation by 100 μm ATP, 10 μm isoproterenol (ISO), 1 μm PGE₁, 500 μm 8-Br-cAMP or 500 μm 8-Br-cGMP applied to the basal (bas) and the apical (ap) surface of C7–MDCK cell monolayers.

Figure 2. Kinetics of protein phosphorylation detected by western blotting

Western blotting was carried out with antibodies against phosphorylated PKA-substrates containing PKA phosphorylation motif (RRXS*/T*) (A) and anti-VASP (B) antibodies in C7–MDCK cells treated with 10 μm isoproterenol (ISO), 1 μm PGE₁ and 100 μm ATP.

Figure 3. Kinetics of modulation of the VASP∼P/VASP ratio by 10 μm isoproterenol (ISO), 1 μm PGE₁ and 100 μm ATP

Mean values obtained from three experiments are shown. The VASP∼P/VASP ratio in the absence of any stimuli was taken as 1.0.

Figure 4. Effect of PKA inhibitor on VASP phosphorylation and I_SC in C7–MDCK cells

A, representative blot showing the effects of AdPKI (3 × 10⁹ v.p. ml⁻¹) and H-89 (10 μm) on VASP phosphorylation in control cells (CON) and cells treated for 2 min with 100 μm ATP, 10 μm isoproterenol (ISO) or 1 μm PGE₁. B, the effect of AdPKI and H-89 on the VASP∼P/VASP ratio and C, maximal values of I_SC (I_SC-MAX) triggered by ATP, isoproterenol and PGE₁. AdPKI and H-89 were applied, respectively, 24 h and 20 min before addition of the above-listed stimuli. Means ± s.e.m. from three (VASP phosphorylation) and four (I_SC) experiments are shown. The VASP∼P/VASP ratio and I_SC-MAX values obtained with ATP-, isoproterenol- or PGE₁-treated cells in the absence of AdPKI and H-89 were taken as 100%.

Figure 5. Effect of AACOCF₃ and AACOCH₃ on PLA₂ activity (A) and I_SC in monolayers of C7–MDCK cells (B)

AACOCF₃ and AACOCH₃ were added at concentrations of 200 μm 30 min before stimulation of cells with 100 μm ATP. Radioactivity of AA spots in the absence of any additions (dpm (mg protein)^-1) was taken as 100%. The maximal values of I_SC developed in ATP-treated cells are shown. Means ± s.e.m. from experiments performed in guadruplicate (PLA₂ activity) or from three experiments (I_SC) are given.

Figure 6. Dose-dependent inhibition by indomethacin of cAMP production (A) and maximal increment of I_SC (B) triggered by 100 μM ATP

Cells were preincubated with indomethacin 20 min before ATP addition. In the absence of ATP, cAMP production varied in the range from 6–10 pmol (mg protein)⁻¹ h⁻¹ and was not affected by indomethacin. I_SC-MAX values in the absence of indomethacin were taken as 100%. Means ± s.e.m. from experiments performed in quadruplicate (cAMP) or from three experiments (I_SC) are given.

Figure 7. Effect of indomethacin (IND) on VASP phosphorylation (A) and transepithelial current (B) in control C7–MDCK cells (CON) and in cells treated with ATP, isoproterenol (ISO) or PGE₁

Indomethacin (10 μm) was added 20 min before 100 μm ATP; 10 μm isoproterenol and 1 μm PGE₁ were applied for the next 2 (A) or 10 (B) min. Means ± s.e.m. from three experiments are shown.

Figure 8. Effect of arachidonic acid (AA) on transepithelial ion current and VASP phosphorylation in C7–MDCK cells

A, typical record of I_SC triggered by basal (bas) and apical (ap) application of AA, linoleic acid (LA) and oleic acid (OA) at a concentration of 10 μm. B, effect of indomethacin (IND, 10 μm), AdPKI (30 v.p. ml⁻¹) and H-89 (10 μm) on VASP phosphorylation in the absence and presence of 10 μm AA. Cells were pretreated for 24 h with AdPKI and for 20 min with indomethacin or H-89 and then AA was applied for 2 min. C, effect of indomethacin, AdPKI and H-89 on maximal I_SC values triggered by the addition of 10 mm AA. I_SC-MAX values obtained in AA-treated cells in the absence of the above-listed inhibitors were taken as 100%. Means ± s.e.m. from three experiments are shown.

Figure 9. Effect of 100 μM ATP, 10 μM indomethacin (IND), 1 μM ionomycin (ION) and 0.5 μM thapsigargin (TG) on [Ca²⁺]_i in control and BAPTA-loaded C7–MDCK cells

BAPTA-AM was added at a concentration of 25 μm simultaneously with fura-2AM. For more details, see Methods.

Figure 10. Typical records of I_SC triggered by the addition of 100 μM ATP and its modulation by 0.5 μM thapsigargin (TG), 1 μM ionomycin (ION) and 25 μM BAPTA-AM

Thapsigargin, ionomycin and BAPTA were added to both serosal and mucosal medium, whereas ATP was applied to the basolateral surface of C7–MDCK monolayers. BAPTA-AM was added 30 min before stimulation with ATP.

Figure 11. P_2Y receptor gene expression in C7–MDCK cells

Real-time PCR was performed using P_2Y subtype-specific primer pairs in conjunction with SYBR Green dye I. mRNA abundance was normalized to GADPH expression and presented relative to P_2Y2 receptors. Means ± s.e.m. from experiments performed in triplicate are shown.

Figure 12. Dose-dependent effect of P_2Y agonists on VASP phosphorylation (A and B), I_SC (C) and intracellular Ca²⁺ concentration (D) in C7–MDCK cells

The VASP∼P/VASP ratio in the absence of any stimuli was taken as 1.0. The maximal increments of I_SC and [Ca²⁺]_i triggered by 2MeSATP and UTP, respectively, were taken as 100%. Mean values obtained from three experiments are shown.

Figure 13. Possible mechanism of nucleotide (P_2Y1) receptor-induced Cl⁻ secretion in monolayers of C7–MDCK cells

1, Cl⁻ channels; AC, adenylyl cyclase; EP, PG_E receptors; PL, AA-containing phospholipids; INDO, indomethacin; a.m and b.m., apical and basolateral membrane, respectively; ?, unknown steps or intermediates; → and —|, activatory and inhibitory stimuli, respectively. Compounds used to verify this model appear in italics. For other abbreviations and details, see text.