Differential Activation of Protein Kinase C Q! Is Associated with Arachidonate Release in Madin-Darby Canine Kidney Cells*

The heterogeneity of the protein kinase C (PKC) gene family strongly suggests that different isoforms may have distinct functions in mediating signal transduc- there very little

it, we report that differential activation (translocation) of PKC (Y is associated with AA release in MDCK cells and that specific downregulation of PKC (Y is associated with a loss of AA release in response to stimulation with dioctanoylglycerol and phorbol ester. We also demonstrate that bradykinin-stimulated AA release was associated with differential activation of PKC a and was inhibited in PKC (Y down-regulated cells. Thus, we conclude that the PKC a isoform is likely to be responsible for mediating AA release in these cells. Protein kinase C (PKC)' is a major component of transmembrane signaling systems (for review see Ref. 1). Agoniststimulated phospholipid hydrolysis produces diacylglycerol, which activates the enzyme by promoting its association with the cell membrane. Tumor-promoting phorbol esters such as phorbol12-myristate 13-acetate and phorbol11,12-dibutyrate (PMA and PDBu, respectively) can substitute for diacylglycerol as activators of the enzyme. The cellular response to such phorbol esters is biphasic. The initial response involves translocation and activation of protein kinase C (2); however, prolonged activation of the enzyme results in its down-regu-  @, and T (III, II, and I, respectively) show different tissue and subcellular distribution and vary in their diacylglycerol, fatty acid, and calcium requirement (reviewed in Refs. 4 and 5). Additionally, differential susceptibility of the isoforms to proteolysis and to phorbol ester-induced down-regulation has been demonstrated in purified preparations of the enzymes (6, 7) and in several cell types including basophilic leukemia cells (6), KM3 cells (8), and BC3H-1 myocytes (9). PKC LY, /l, and 7 are the products of distinct genes, and subspecies of PKC p(pl and &) are derived from alternative splicing of a single gene (see Refs. 4 and 5 for reviews). The heterogeneity of the protein kinase C gene family suggests that different isoforms may have distinct functions within a given tissue or subcellular compartment.
In these experiments we sought to identify the isoforms of PKC present in the clonally derived renal epithelial cell line MDCK-Dl (10, 11) and to determine their relative susceptibility to down-regulation and their role in mediating a cellular response, namely arachidonate release. PKC-stimulated arachidonate release has been demonstrated in a variety of cell types (12-15) including MDCK cells (16)(17)(18)(19)(20) in response to both phorbol ester and hormonal stimulation.
Here we report that (

AND DISCUSSION
Activation of PKC is associated with translocation of the enzyme from cytosolic to membrane compartments; such translocation occurs in response to agonist-stimulated diacyiglycerol production, and this activation process can be mimicked by tumor-promoting phorbol esters and cell permeant diacylglycerol analogues such as diC8. When we treated MDCK cells with 100 nM PMA or 200 PM diC8 for 30 min we observed increased activity of the enzyme in the particulate fraction ( Fig. 1) (1, 3), and accompanying this activation of the enzyme was an enhancement in arachidonate release (see control values, Fig. 2A). Treatment of the cells with 1 PM BK caused a transient increase in PKC activity in the particulate fraction; this was detectable as early as 30 s after the addition of BK and had declined to basal levels by 5 min (data not shown). The kinetics of this translocation were similar to those observed for cY1-adrenergic activation of PKC in these cells (24).
To determine the relative involvement of PKC isoforms in these processes we used isoform-specific antibodies in immunoblotting procedures. Using anti-a, -& and -7 antibodies we identified PKC types (Y and p in MDCK cells; no 7 isoform was detectable.' These observations are consistent with the known tissue-specific distribution of the isoforms (4, 5), the 7 form being found almost exclusively in the nervous system and the cy and @ forms being found in peripheral tissues in varying amounts. The specificity of the isoform-specific antisera used in these experiments has previously been documented (21). Treatment of the cells with PMA caused selective translocation of PKC 01, while the relative distribution of PKC @ was unchanged by PMA treatment (Fig. 3A). BK treatment of the cells also stimulated PKC (Y translocation after 1 min (Fig. 3B); in parallel experiments translocation of PKC firI, the predominant /3 isoform in these cells, was not observed. Treatment of the cells with diC8 also resulted in translocation of PKC (Y (data not shown). A causative relationship between PKC cr activation and arachidonate release is suggested by the fact that PKC (Y translocation is observed prior to or concomitant with phorbol ester (18)  of PKC 01 in response to short term PMA treatment, we observed that 18-h treatment of the cells with 100 nM PDBu resulted in selective down-regulation of PKC LY (Fig. 4). Moreover, down-regulation of PKC in response to prolonged stimulation with phorbol ester resulted in inhibition of arachidonate release stimulated by PMA, BK, and diC8 (Fig. 2). In rat basophilic leukemia cells (8) and in KM-3 cells (7) PKC /3 has been shown to be most susceptible to proteolysis whereas in BCSH-1 myocytes PKC (Y is more susceptible (9).
To verify that the decrease in immunoreactive PKC (Y which we observed is associated with decreased activity of the enzyme was assayed PKC activity in lysates of cells treated with 100 nM PDBu or 100 nM 4~PDD (an inactive phorbol ester) or 0.1% ethanol (control).
Overnight treatment with PDBu resulted in a decrease of PKC activity from 1.58 f 0.3 to 0.73 + 0.2 pmol of "'P incorporated per pg of protein per min (mean + SE. of four independent experiments). Overnight treatment with 4a-PDD did not significantly alter PKC activity in the lysates. To eliminate the possibility that the presence of PDBu in down-regulated cell lysates might compromise the ability of the added diolein to stimulate PKC and epinephrine all promote deacylation of membrane phospholipids by phospholipase AY (18,19). The mechanism whereby PKC (Y activates phospholipase AZ might involve activation of the enzyme by phosphorylation (13,17), regulation of PLA, inhibitory proteins such as the lipocortins (33), or stimulatory proteins such as phospholipase A2 activating protein (34). Alteration of transmembrane calcium flux may also be involved (35). The inability of down-regulation of PKC 01 to completely abolish lyso-PE production stimulated by bradykinin suggests the possibility that BK may modulate phospholipase A, through additional mechanisms other than PKC activation.
Possible mechanisms are direct activation of phospholipase AS consequent to bradykinininduced calcium influx (36) or coupling of bradykinin receptors to phospholipase A2 through a guanine nucleotide binding protein (37).
In conclusion, the data presented here provide the first evidence of a role for PKC (Y but not p in phorbol esterstimulated arachidonate release as under acute conditions where: (i) PMA stimulates arachidonate release in these cells PKC (Y but not PKC fi is activated (translocated); (ii) BK stimulates arachidonate release in these cells PKC CY but not pii is translocated; and (iii) down-regulation of PKC 01 but not PKC fi completely inhibits arachidonate release stimulated by PMA, bradykinin, and the diacylglycerol analog diC8. We speculate that arachidonate release is likely to be one of many cellular responses that can be selectively regulated by PKC isoforms.