PKCbetaI mediates the inhibition of P2Y receptor-induced inositol phosphate formation in endothelial cells

Abstract

1. Bovine pulmonary artery endothelium (CPAE) expresses phospholipase C (PLC)-linked P2Y1 and P2Y2 receptors, for them 2-methylthio-ATP (2MeSATP) and UTP are respective agonists. Here, we have investigated the particular protein kinase C (PKC) isoform(s) responsible for the inhibition of P2Y1 and P2Y2 receptor-evoked inositol phosphate (IP) formation by phorbol 12-myristate 13-acetate (PMA). 2. Although short-term (20 min) pretreatment of cells with PMA attenuated 2MeSATP- and UTP-induced phosphoinositide (PI) breakdown, this inhibition was lost after 15 h. Preincubation with PMA for 24 h, on the contrary, potentiated 2MeSATP and UTP responses. The IP formation stimulated by NaF was unaltered by PMA pretreatment. 3. Western blot analysis showed that treatment of CPAE with PMA resulted in a rapid translocation of PKC isoform betaI, epsilon and mu, but not lambda, from the cytosol to the membrane fraction. 4. Pretreatment of the selective PKC inhibitor Ro 31-8220 attenuated the inhibitory effect of PMA on IP formation. Go 6976 (an inhibitor of conventional PKCalpha, beta and gamma) and LY 379196 (a selective PKCbeta inhibitor) also dose-dependently inhibited the PMA-mediated desensitization. 5. Transfection of PKCbeta-specific antisense oligonucleotide reduced PKCbetaI protein level and inhibited PMA-mediated PI reduction. 6. RT - PCR analysis showed that PMA treatment for 4 - 24 h up-regulated P2Y1 and P2Y2 receptors at the mRNA levels. 7. These results suggest that PKCbetaI may exert a negative feedback regulation on endothelial P2Y1 and P2Y2 receptor-mediated PI turnover. The down-regulation of PKCbetaI and enhanced P2Y receptor expression together might contribute to the late PI enhancing effect of PMA.

Figure 1

Time-dependent effects of PMA on 2MeSATP- and UTP-induced [³H]-IP formation in CPAE cells. Cells were pretreated with PMA (1 μM) for different periods of time before challenge with 100 μM 2MeSATP (□) or 100 μM UTP (⋄). Results are expressed as the mean±s.e.mean of three independent experiments. ^*Using ANOVA followed by Dunnetts test, P<0.05 was considered significant as compared to the 2MeSATP- or UTP-induced PI turnover without PMA pretreatment.

Figure 2

Effect of PMA treatment on the translocation of PKCβI, ε, λ and μ in CPAE cells. Cells were treated with 1 μM PMA for different time (2, 5, and 10 min) and then fractionated into the cytosolic and membrane fractions. Equal amount of protein was separated by 9% SDS–PAGE, transferred to nitrocellulose membrane, and immunoblotted with antibodies specific for PKCβI, ε, λ and μ, as described in ‘Methods'. The values in the parentheses indicate the changes of PKC immunoreactivities which were expressed as percentages of control. The results are representative of three experiments.

Figure 3

Concentration-dependent effects of Go 6976 and LY 379196 on PMA-induced inhibition of 2MeSATP- and UTP-evoked PI turnover in CPAE cells. In (a) and (b), cells were incubated with vehicle (DMSO) or Go 6976 (100 nM–1 μM) for 20 min followed by addition of PMA at concentrations within 0.3–100 nM for another 30 min. Subsequent to these treatments, 2MeSATP (100 μM) (a) or UTP (100 μM) (b) was added to trigger the accumulation of [³H]-IP. In (c), cells were pretreated with 30 or 100 nM LY 379196 for 20 min followed by addition of 1 μM PMA for another 30 min. Then 2MeSATP (100 μM) or UTP (100 μM) was added to induce PI turnover. The data represent the mean±s.e.mean of three independent experiments performed in duplicate. For (a) and (b), using two-way ANOVA, Go 6976 caused a significant difference from control (P<0.0001). When significance was reached, using Dunnetts test, ^*P<0.05 that are significantly different from PMA response in the absence of Go 6976. For (c), using ANOVA followed by Dunnetts test, ^*P<0.05 was considered significant as compared to the PMA response in the absence of PKC inhibitors.

Figure 4

Antisense oligonucleotide inhibits immunoreactive PKCβI protein expression and PMA-induced reduction of 2MeSATP- and UTP-induced PI turnover. (a) Cells were treated with vehicle, antisense or sense PKCβ oligonucleotide (1.5 μg) for 4 h, then incubated in replaced growth medium for another 48 h as described under ‘Methods'. The immunoreactivities of PKC isoforms were analysed by immunoblots and the results shown are representative of three experiments. (b) After cells were processed as described in (a), PMA (1 μM) was incubated for 20 min followed by the stimulation with 2MeSATP (100 μM) or UTP (100 μM) for 30 min. The accumulated [³H]-IP was measured. The data represent the mean±s.e.mean of three independent experiments performed in duplicate. Using ANOVA followed by Dunnetts test, ^*significantly different (P<0.05) from the control response. ^**Significantly different (P<0.05) from the inhibitory PMA response without oligonucleotide pretreatment.

Figure 5

RT–PCR analysis of P2Y₁ and P2Y₂ mRNA levels in PMA-treated cells. CPAE cells were treated with 100 nM PMA for 4, 8, 18 or 24 h followed by extraction of total RNA and analysis of mRNA levels of P2Y₁, P2Y₂ receptor and β-actin. RT–PCR technique was performed as described in ‘Methods'. β-actin levels normalized the amount of cDNA template used in each PCR reaction. The results are representative of three experiments.