Differential coupling of the human P2Y(11) receptor to phospholipase C and adenylyl cyclase

Abstract

1. The human P2Y(11) (hP2Y(11)) receptor was stably expressed in two cell lines, 1321N1 human astrocytoma cells (1321N1-hP2Y(11)) and Chinese hamster ovary cells (CHO-hP2Y(11)), and its coupling to phospholipase C and adenylyl cyclase was assessed. 2. In 1321N1-hP2Y(11) cells, ATP promoted inositol phosphate (IP) accumulation with low microM potency (EC(50)=8.5+/-0.1 microM), whereas it was 15 fold less potent (130+/-10 microM) in evoking cyclic AMP production. 3. In CHO-hP2Y(11) cells, ATP promoted IP accumulation with slightly higher potency (EC(50)=3.6+/-1.3 microM) than in 1321N1-hP2Y(11) cells, but it was still 15 fold less potent in promoting cyclic AMP accumulation (EC(50)=62.4+/-15.6 microM) than for IP accumulation. Comparable differences in potencies for promoting the two second messenger responses were observed with other adenosine nucleotide analogues. 4. In 1321N1-hP2Y(11) and CHO-hP2Y(11) cells, down regulation of PKC by chronic treatment with phorbol ester decreased ATP-promoted cyclic AMP accumulation by 60--80% (P<0.001) with no change in its potency. Likewise, chelation of intracellular Ca(2+) decreased ATP-promoted cyclic AMP accumulation by approximately 45% in 1321N1-hP2Y(11) cells, whereas chelation had no effect on either the efficacy or potency of ATP in CHO-hP2Y(11) cells. 5. We conclude that coupling of hP2Y(11) receptors to adenylyl cyclase in these cell lines is much weaker than coupling to phospholipase C, and that activation of PKC and intracellular Ca(2+) mobilization as consequences of inositol lipid hydrolysis potentiates the capacity of ATP to increase cyclic AMP accumulation in both 1321N1-hP2Y(11) and CHO-hP2Y(11) cells.

Figure 1

Concentration-response curves of ATP and ADP for increasing IP and cyclic AMP levels in 1321N1-hP2Y₁₁ cells. Each point is the mean response from three experiments. Vertical lines indicate s.d. mean.

Figure 2

Concentration-response curves of ATP and ADP for increasing IP and cyclic AMP levels in CHO-hP2Y₁₁ cells. (A) Concentration-response curves for ATP-promoted IP production in CHO-K1 cells infected with empty retrovirus (pLXSN) or with retrovirus harbouring hP2Y₁₁ receptor cDNA (P2Y₁₁). (B) Concentration-response curves of ATP and ADP for increasing IP and cyclic AMP levels in CHO-hP2Y₁₁ cells. Each point is the mean response from five experiments. Vertical lines indicate s.d. mean.

Figure 3

Effects of Ca²⁺ chelation and PKC down-regulation on ATP-promoted cyclic AMP accumulation in 1321N1-hP2Y₁₁ cells. (A) Cells treated with BAPTA-AM (50 μM) for 10 min, PMA (1 μM) for 20 h, or a combination of both were challenged with buffer or 300 μM ATP and cyclic AMP accumulation was measured as described in Methods. (B) 1321N1 cells expressing the hD1 dopamine receptor were treated as described in (A), challenged with 100 μM dopamine for 10 min, and cyclic AMP accumulation was measured. (C) Concentration-response curves of ATP for promotion of cyclic AMP accumulation in vehicle- or PMA- (1 μM for 20 h) plus BAPTA-AM- (50 μM for 10 min) treated 1321N1-hP2Y₁₁ cells. Data shown are the mean±s.d. of triplicate assays from a representative experiment repeated three times. ^**P⩽0.01 and ^***P⩽0.001 relative to control response. ^‡‡P⩽0.01 relative to PMA treatment alone.

Figure 4

Effects of Ca²⁺ chelation and PKC down-regulation on ATP-promoted cyclic AMP accumulation in CHO-hP2Y₁₁ cells. (A) Cells treated with BAPTA-AM (50 μM) for 10 min, PMA (1 μM) for 20 h, or a combination of both were challenged with buffer, 300 μM ATP, or 10 μM PGE₁ for 10 min and cyclic AMP accumulation was measured as described in Methods. (B) Concentration-response curves of ATP for promotion of cyclic AMP accumulation in untreated or PMA- (1 μM for 20 h) and BAPTA-AM-treated (50 μM for 10 min) CHO-hP2Y₁₁ cells. Data shown are the mean±s.d. of triplicate assays from a representative experiment repeated three times. ^**P⩽0.01 relative to control responses.

Figure 5

Effects of acute PKC activation and intracellular Ca²⁺ mobilization on cyclic AMP accumulation in CHO-hP2Y₁₁ cells and 1321N1-hD1 cells. (A) CHO-hP2Y₁₁ cells were treated with either vehicle or PMA (1 μM) for 10 min, and cyclic AMP accumulation was measured following challenge with buffer, 300 μM ATP, 10 μM PGE₁ or 30 μM forskolin for an additional 10 min. ^*P⩽0.05, ^**P⩽0.01 relative to untreated responses. (B) 1321N1-hD1 cells were challenged with either vehicle or dopamine (100 μM) for 10 min in the presence or absence of carbachol (300 μM) and cyclic AMP accumulation was measured. Data shown are the mean±s.d. of triplicate assays from a representative experiment repeated three times. ^***P⩽0.001 relative to dopamine alone.