Mingzhao Xing

Protein kinase C-dependent activation of cytosolic phospholipase A2 and mitogen-activated protein kinase by alpha 1-adrenergic receptors in Madin-Darby canine kidney cells.

We have characterized the mechanism whereby a G protein-coupled receptor, the alpha 1-adrenergic receptor, promotes cellular AA release via the activation of phospholipase A2 (PLA2) in Madin-Darby canine kidney (MDCK-D1) cells. Stimulation of cells with the receptor agonist epinephrine or with the protein kinase C (PKC) activator PMA increased AA release in intact cells and the activity of PLA2 in subsequently prepared cell lysates. The effects of epinephrine were mediated by alpha 1-adrenergic receptors since they were blocked by the alpha 1-adrenergic antagonist prazosin. Epinephrine- and PMA-promoted AA release and activation of the PLA2 were inhibited by AACOCF3, an inhibitor of the 85-kD cPLA2. The 85-kD cPLA2 could be immunoprecipitated from the cell lysate using a specific anti-cPLA2 serum. Enhanced cPLA2 activity in cells treated with epinephrine or PMA could be recovered in such immunoprecipitates, thus directly demonstrating that alpha 1-adrenergic receptors activate the 85-kD cPLA2. Activation of cPLA2 in cell lysates by PMA or epinephrine could be reversed by treatment of lysates with exogenous phosphatase. In addition, both PMA and epinephrine induced a molecular weight shift, consistent with phosphorylation, as well as an increase in activity of mitogen-activated protein (MAP) kinase. The time course of epinephrine-promoted activation of MAP kinase preceded that of the accumulation of released AA and correlated with the time course of cPLA2 activation. Down-regulation of PKC by overnight incubation of cells with PMA or inhibition of PKC with the PKC inhibitor sphingosine blocked the stimulation of MAP kinase by epinephrine and, correspondingly, epinephrine-promoted AA release was inhibited under these conditions. Similarly, blockade of MAP kinase stimulation by the MAP kinase cascade inhibitor PD098059 inhibited epinephrine-promoted AA release. The sensitivity to Ca2+ was similar, although the maximal activity of cPLA2 was enhanced by treatment of cells with epinephrine or PMA. The data thus demonstrate that in MDCK-D1 cells alpha 1-adrenergic receptors regulate AA release through phosphorylation-dependent activation of the 85-kD cPLA2 by MAP kinase subsequent to activation of PKC. This may represent a general mechanism by which G protein-coupled receptors stimulate AA release and formation of products of AA metabolism.

Dual role of protein kinase C in the regulation of cPLA2-mediated arachidonic acid release by P2U receptors in MDCK-D1 cells: involvement of MAP kinase-dependent and -independent pathways.

Defining the mechanism for regulation of arachidonic acid (AA) release is important for understanding cellular production of AA metabolites, such as prostaglandins and leukotrienes. Here we have investigated the differential roles of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase in the regulation of cytosolic phospholipase A2 (cPLA2)-mediated AA release by P2U-purinergic receptors in MDCK-D1 cells. Treatment of cells with the P2U receptor agonists ATP and UTP increased PLA2 activity in subsequently prepared cell lysates. PLA2 activity was inhibited by the cPLA2 inhibitor AACOCF3, as was AA release in intact cells. Increased PLA2 activity was recovered in anti-cPLA2 immunoprecipitates of lysates derived from nucleotide-treated cells, and was lost from the immunodepleted lysates. Thus, cPLA2 is responsible for AA release by P2U receptors in MDCK-D1 cells. P2U receptors also activated MAP kinase. This activation was PKC-dependent since phorbol 12-myristate 13-acetate (PMA) promoted down-regulation of PKC-eliminated MAP kinase activation by ATP or UTP. Treatment of cells with the MAP kinase cascade inhibitor PD098059, the PKC inhibitor GF109203X, or down-regulation of PKC by PMA treatment, all suppressed AA release promoted by ATP or UTP, suggesting that both MAP kinase and PKC are involved in the regulation of cPLA2 by P2U receptors. Differential effects of GF109203X on cPLA2-mediated AA release and MAP kinase activation, however, were observed: at low concentrations, GF109203X inhibited AA release promoted by ATP, UTP, or PMA without affecting MAP kinase activation. Since GF109203X is more selective for PKCalpha, PKCalpha may act independently of MAP kinase to regulate cPLA2 in MDCK-D1 cells. This conclusion is further supported by data showing that PMA-promoted AA release, but not MAP kinase activation, was suppressed in cells in which PKCalpha expression was decreased by antisense transfection. Based on these data, we propose a model whereby both MAP kinase and PKC are required for cPLA2-mediated AA release by P2U receptors in MDCK-D1 cells. PKC plays a dual role in this process through the utilization of different isoforms: PKCalpha regulates cPLA2-mediated AA release independently of MAP kinase, while other PKC isoforms act through MAP kinase activation. This model contrasts with our recently demonstrated mechanism (J. Clin. Invest. 99:1302-1310.) whereby alpha1-adrenergic receptors in the same cell type regulate cPLA2-mediated AA release only through sequential activation of PKC and MAP kinase.

Extracellular ATP and cAMP as Paracrine and Interorgan Regulators of Renal Function P2Y Receptors of MDCK Cells: Epithelial Cell Regulation by Extracellular Nucleotides

1. Madin–Darby canine kidney (MDCK) cells, a well‐ differentiated renal epithelial cell line derived from distal tubule/collecting duct, respond to extracellular nucleotides by altering ion flux and the production of arachidonic acid‐derived products, in particular prostaglandin E2 (PGE2). Our work has defined the receptors and signalling events involved in such responses.

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.