Inhibition of Phospholipase A2-mediated Arachidonic Acid Release by Cyclic AMP Defines a Negative Feedback Loop for P2Y Receptor Activation in Madin-Darby Canine Kidney D1 Cells*

In Madin-Darby canine kidney D1cells extracellular nucleotides activate P2Y receptors that couple to several signal transduction pathways, including stimulation of multiple phospholipases and adenylyl cyclase. For one class of P2Y receptors, P2Y2 receptors, this stimulation of adenylyl cyclase and increase in cAMP occurs via the conversion of phospholipase A2 (PLA2)-generated arachidonic acid (AA) to prostaglandins (e.g. PGE2). These prostaglandins then stimulate adenylyl cyclase activity, presumably via activation of prostanoid receptors. In the current study we show that agents that increase cellular cAMP levels (including PGE2, forskolin, and the β-adrenergic agonist isoproterenol) can inhibit P2Y receptor-promoted AA release. The protein kinase A (PKA) inhibitor H89 blocks this effect, suggesting that this feedback inhibition occurs via activation of PKA. Studies with PGE2indicate that inhibition of AA release is attributable to inhibition of mitogen-activated protein kinase activity and in turn of P2Y receptor stimulated PLA2 activity. Although cAMP/PKA-mediated inhibition occurs for P2Yreceptor-promoted AA release, we did not find such inhibition for epinephrine (α1-adrenergic) or bradykinin-mediated AA release. Taken together, these results indicate that negative feedback regulation via cAMP/PKA-mediated inhibition of mitogen-activated protein kinase occurs for some, but not all, classes of receptors that promote PLA2 activation and AA release. We speculate that receptor-selective feedback inhibition occurs because PLA2activation by different receptors in Madin-Darby canine kidney D1 cells involves the utilization of different signaling components that are differentially sensitive to increases in cAMP or, alternatively, because of compartmentation of signaling components.

Arachidonic acid (AA) 1 and its eicosanoid metabolites (e.g. prostaglandins and leukotrienes) play critical roles in the initiation or modulation of a broad spectrum of biological responses, including many inflammatory processes. In mammalian cells AA is normally stored in membrane phospholipids and released primarily by phospholipase A 2 (PLA 2 ). Among several types of mammalian PLA 2 s, the 85-kDa cytosolic form (cPLA 2 ) appears to specifically catalyze receptor-promoted AA release. Considerable efforts have been made in recent years to study the mechanism for the activation of cPLA 2 and the subsequent release of AA. In a variety of cell types, cPLA 2 activation occurs as a result of phosphorylation by mitogen-activated protein (MAP) kinase (1).
We have recently demonstrated that in MDCK-D 1 cells P 2Y2 (previously termed P 2U ) purinergic receptor-promoted AA release is mediated through cPLA 2 activation by MAP kinase (2). Other data have shown that stimulation of P 2Y2 purinergic receptors in these cells also stimulates adenylyl cyclase (AC) activity, increasing cellular cAMP levels through an autocrine/ paracrine mechanism involving prostaglandin E 2 (PGE 2 ) production subsequent to cPLA 2 -mediated AA release (3,4). In the present study we sought to assess the relationship of these two signaling pathways activated by P 2Y2 purinergic receptors. We found that activation of the AC system can inhibit cPLA 2mediated AA release by P 2Y2 purinergic receptors through the inhibition of MAP kinase in MDCK-D 1 cells. Therefore, we define a negative feedback mechanism via an autocrine/paracrine cycle in which P 2Y2 receptors can attenuate the activation of PLA 2 and AA release initiated by receptor agonists.

EXPERIMENTAL PROCEDURES
Cell Culture-MDCK-D 1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% serum and passaged every 3-4 days by trypsinization using trypsin/EDTA. Cells were used for experiments when grown to approximately 70% confluence.
Assay of cAMP-Growth medium was removed from cells, and cells were equilibrated for 30 min at 37°C in serum-free Dulbecco's modified Eagle's medium containing 20 mM Hepes buffer (DMEH, pH 7.4). Subsequently cells were incubated in fresh DMEH with PGE 2 for 5 min at 37°C in the presence of 200 M isobutylmethylxanthine or 100 M Ro20 -1724, two different phosphodiesterase inhibitors. Reactions were terminated by aspiration of medium and addition of 7.5% trichloroacetic acid. Intracellular cAMP levels were determined by radioimmunoassay (Calbiochem, CA) of trichloroacetic acid extracts following acetylation, as described previously (3).
[ 3  Cells were washed four times with DMEH supplemented with 5 mg/ml bovine serum albumin and then incubated in the same medium at 37°C for 15-20 min to equilibrate the temperature. Agents of interest were then added in 1 ml of 37°C medium after removing equilibration medium. Release of [ 3 H]AA was assayed and normalized to the percentage of incorporated radioactivity, as described previously (2). cPLA 2 Activity Assay of Cell Lysates-cPLA 2 activity was assayed in lysates prepared from cells incubated with various agents, as described previously (2). Briefly, cells were incubated with indicated agonists in DMEH for 10 min at 37°C, washed with ice-cold buffer containing 250 mM sucrose, 50 mM Hepes, pH 7.4, 1 mM EGTA, 1 mM EDTA, phosphatase inhibitors (200 M Na 3 VO 4 , 1 mM levamisole) and protease inhibitors (500 M phenylmethylsulfonyl fluoride, 8 M pepstatin, 16 M * This work was supported by National Institutes of Health Grants GM31987 and HL35847, by National Research Service Award training grants, and by a NRSA fellowship (to M. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Assay of Phospholipase D Activation-MDCK-D 1 cells were labeled by an overnight incubation with [ 3 H] palmitate. Washed cells were then incubated for 1 h with 20 M indomethacin (to block PGE 2 formation), with 0.5 M isobutylmethylxanthine for 20 min, with 20 M PGE 2 or 50 M isoproterenol for 20 min, and then with either 300 M ATP or 300 M UTP for 10 min. Cells were lysed and phosphatidylethanol was resolved by thin layer chromatography, as described previously (5). Phosphatidylethanol was expressed as the percentage of total cellular radioactivity.
Phosphorylation-induced Mobility Shift, SDS-Polyacrylamide Gel Electrophoresis, and Immunoblotting of MAP Kinase-Cells were washed five times with DMEH supplemented with 2 mg/ml bovine serum albumin, incubated in this medium at 37°C for 1 h, and then with specified agonists for 3 min. Reactions were stopped by aspiration of medium and washing of cells four times with ice-cold buffer (62.5 mM Tris HCI, pH 6.8, plus 10% glycerol), and protease and phosphatase inhibitors were used for PLA 2 activity assays. Scraped cells were lysed in SDS-polyacrylamide gel electrophoresis loading buffer and boiled for five min, and samples were electrophoresed on SDS-polyacrylamide gel electrophoresis using 7.5% acrylamide. Following transfer to an Immobilon-P polyvinylidene fluoride membrane (Millipore) and blocking for 1 h with 5% nonfat dry milk dissolved in phosphate-buffered saline, membranes were incubated with 1:2000 -3000 diluted anti-p42 MAP kinase rabbit serum for 90 min and then with 1:2000 diluted horseradish-peroxidase-linked donkey anti-rabbit immunoglobulin for 1 h (both in 5% nonfat dry milk dissolved in phosphate-buffered saline) and then washed four times with phosphate-buffered saline (5 min each wash). MAP kinase bands were visualized using ECL immunoblotting detection reagents (Amersham Pharmacia Biotech).
Data Presentation-Unless otherwise specified, the data shown in figures are the means Ϯ S.D. of triplicate measurements and are representative of results obtained in two to four experiments. Results were analyzed for statistical significance by one-way analysis of variance (with Bonferroni's correction, where appropriate).

Increase in Cellular cAMP Inhibits P 2Y2
Receptor-promoted AA Release and cPLA 2 Activation in MDCK-D 1 Cells-We have previously demonstrated in MDCK-D 1 cells that activation of P 2Y2 purinergic receptors results in AA release via activation of cPLA 2 (2) and that stimulation of P 2Y2 purinergic receptors in these cells can increase cellular cAMP levels via the action of the PGE 2 generated from AA (3,4). To further investigate the relationship of these two signaling pathways activated by P 2Y2 receptors, we examined the effects of cAMP-increasing agents on P 2Y2 receptor-promoted AA release. As shown in Fig. 1, agents that increase cAMP in MDCK-D 1 cells (PGE 2 , forskolin, and Ro20 -1724) inhibited AA release stimulated by the P 2 receptor agonists ATP and UTP. Isoproterenol also inhibited the AA release stimulated by these purinergic agonists (data not shown). PGE 2 -mediated stimulation of cAMP and inhibition of P 2Y2 receptor-promoted AA release displayed a similar concentration-response relationship, with a nearly maximal effect of PGE 2 achieved at 1-10 M both for production of cAMP and inhibition of AA release (Fig. 2). Because P 2Y2 receptor-promoted AA release in MDCK-D 1 cells is mediated by activation of cPLA 2 (2), we tested whether an increase in cAMP would blunt activation of this lipase. Indeed, as shown in Fig. 3, activation of PLA 2 activity in cell lysates by the specific P 2Y2 agonist UTP was substantially inhibited by treatment of cells with PGE 2 . Therefore, increases in cellular cAMP decrease AA release and cPLA 2 activation by P 2Y2 receptors in MDCK-D 1 cells.
MDCK-D 1 cells possess ␣ 1 -adrenergic receptors and B 2 -bradykinin receptors that promote AA release in response to stimulation by epinephrine and bradykinin, respectively (8 -10). As do P 2Y2 receptors, ␣ 1 -adrenergic receptors and bradykinin receptors promote AA release through activation of cPLA 2 in MDCK-D 1 cells (2, 3, 7). Therefore, we tested whether increases in cAMP would regulate AA release by these different receptors. Unlike the results obtained for P 2Y2 receptors, AA release elicited by ␣ 1 -adrenergic receptors and bradykinin receptors was not inhibited by forskolin, Ro20 -1724 (an inhibitor of phosphodiesterase), or both (Fig. 1B). Treatment with PGE 2 alone or with PGE 2 and Ro20 -1724 yielded similar results (data not shown). Neither forskolin nor PGE 2 stimulation of cAMP accumulation was diminished by the presence of various concentrations of epinephrine or bradykinin, as compared with the presence of ATP or UTP (data not shown).
Activation of PKA and Inhibition of MAP Kinase Are Responsible for the cAMP-mediated Inhibition of P 2Y2 Receptor-promoted AA Release-To test whether the inhibitory effect of the increase in cellular cAMP on P 2Y2 receptor-promoted AA release and cPLA 2 activation in MDCK-D 1 cells is mediated by the activation of PKA, we examined the effect of the PKA inhibitor H89 on PGE 2 -mediated inhibition of AA release. As shown in Fig. 4, PGE 2 -mediated inhibition of ATP-or UTPstimulated AA release was completely prevented by pretreatment of cells with H89, whereas H89 had no statistically significant effect on basal or P 2Y2 receptor-promoted AA release. These data suggest that activation of PKA by cAMP is responsible for the inhibitory effects of increased cellular cAMP levels on P 2Y2 receptor-promoted AA release.
Because MAP kinase plays a critical role in the activation of cPLA 2 and AA release in MDCK-D 1 cells (2), we next assessed whether activation of MAP kinase by P 2Y2 receptors was inhibited by pretreatment of cells with PGE 2 . Consistent with this idea were results with epinephrine, which also activates MAP kinase activity in MDCK-D 1 cells (7). PGE 2 blocked the MAP kinase activation by UTP but not that by epinephrine (Fig. 5). As shown in Fig. 6, the UTP-induced gel shift of MAP kinase was inhibited by incubation of cells with PGE 2 . This PGE 2 -mediated inhibition of MAP kinase was reversed by treatment of cells with H89, suggesting that activation of PKA is responsible for both the cAMP-mediated inhibition of MAP kinase activation and inhibition of AA release and cPLA 2 activation. Based on these and previous data related to the role of MAP kinase on P 2Y receptor-promoted activation of cPLA 2 in MDCK-D 1 cells, we conclude that the cAMP/PKA system negatively regulates cPLA 2 activated by P 2Y2 receptors through the inhibition of MAP kinase activation. P 2Y Receptor Activation of Phospholipase D Activity Is Not Inhibited by Elevation of cAMP-In addition to activation cPLA 2 , P 2Y receptors can also increase phospholipase D activity in MDCK-D 1 cells (5). To determine whether the inhibitory effect of cAMP on cPLA 2 activation occurs at more upstream levels of the signaling cascade, such as at the level of receptor or G protein, we measured the effect of increasing cAMP on phospholipase D activity. The inability of increases in cAMP to blunt ATP-and UTP-mediated phospholipase D activity (Fig.  7) argues that a more distal, nonshared component, such as MAP kinase (Figs. 5 and 6), is the site of negative regulation of cPLA 2 /AA release by increases in cAMP. DISCUSSION Inhibitory effects of cAMP-increasing agents on AA release have been observed in several previous studies (e.g. Refs. [11][12][13]. However, the molecular mechanism for this phenomenon was not defined in these earlier studies. We recently showed that P 2Y2 receptors utilize MAP kinase and protein kinase C␣ as parallel pathways for the activation of cPLA 2 in MDCK-D 1 cells; blockade of either of these two pathways impairs the activation of cPLA 2 by P 2Y2 receptor agonists (2). In the present study, we found that activation of the AC/PKA system can inhibit P 2Y2 receptor-promoted cPLA 2 activation by inhibiting the MAP kinase signaling pathway. This finding is consistent with the reports that cAMP and cAMP-mediated activation of PKA can inhibit MAP kinase activation in other types of cells (14 -16).
AA release activated by P 2Y2 receptors, but not activated that by ␣ 1 -adrenergic or B 2 -bradykinin receptors, was inhibited by the AC/PKA system (Fig. 1). Because we have found that MAP kinase appears not to be involved in the regulation of AA release/cPLA 2 activation by bradykinin receptors in MDCK-D 1 cells (6), we were not surprised by the absence of inhibition of bradykinin receptor-promoted AA release by the AC/PKA system. Another group has used MDCK-D 1 cells and observed slight inhibition of bradykinin receptor-promoted AA release by cAMP (17). However, a similar inhibition also was noted of "basal" AA release in that study. The absence of inhibition by the AC/PKA system on ␣ 1 -adrenergic receptor-promoted AA release was unexpected, because MAP kinase activation is responsible for AA release/cPLA 2 activation by ␣ 1 -adrenergic receptors in MDCK-D 1 cells (7). The ability of the AC/PKA system to inhibit P 2Y2 receptor-mediated, but not ␣ 1 -adrenergic receptor-mediated activation of MAP kinase (Fig. 4), leads us to conclude that the differential inhibitory effects of the AC/PKA system on AA release lies in differences in MAP kinase activation by the two receptors. This conclusion is also supported by studies showing a lack of inhibition by cAMP of P 2 receptormediated activation of PLD activity (Fig. 7). Other data indicate that MAP kinase activation is not required for PLD activation by P 2 receptors or ␣ 1 -adrenergic receptors in MDCK cells (18). 2 We speculate that P 2Y2 purinergic and ␣ 1b -adrenergic receptors are coupled to cPLA 2 and MAP kinase through different signaling pathways (perhaps via different G proteins) that are differentially sensitive to increases in cAMP. Alternatively, signaling components utilized by the two types of receptors are compartmentalized such that increases in cAMP selectively regulate components unique to the P 2Y2 receptor pathway.
In conclusion, the present study demonstrates that the AC/PKA system plays a negative role in the regulation of AA release/cPLA 2 activation by P 2Y2 receptors through inhibition of MAP kinase activation. This negative regulation occurs for P 2Y2 receptors but not for two other classes of receptors coupled to cPLA 2 /AA release and is apparently secondary to effects of cAMP/PKA to inhibit MAP kinase activation. Because the P 2Y2 receptor can activate the AC/PKA system by promoting cPLA 2 -mediated release of AA and its subsequent conversion to PGE 2 (3,4), our results define a feedback cycle whereby P 2Y2 receptors in MDCK-D 1 cells activate AA release and production of PGE 2 . PGE 2 , in turn, activates the AC/PKA system and then inhibits MAP kinase to decrease the AA signaling cascade. Such a cycle could serve to produce homologous desensitization of the purinergic receptor pathway in response to nucleotides and thus would blunt ongoing production of AA and AA metabolites. Moreover, the cross-talk that occurs between AC-stimulating pathways and the purinergic pathway also represents a mechanism for the heterologous desensitization of the P 2Y purinergic receptor pathway. The feedback cycle described herein may contribute to both physiologic and pharmacologic regulation of the P 2Y purinergic receptor signaling. Overall, these results, together with evidence that P 2Y2 receptors in MDCK-D 1 cells are coupled to cAMP production via release of AA and its conversion to PGE 2 , define a potentially important feedback loop for regulation of AA formation.