WB: myc
3.3.6 Pharmacological characteristics of the receptor hetero-oligomer
3.3.6.1 Intracellular calcium rise
The hetero-oligomerization of the P2Y11GFP receptor with the endogenous P2Y1 receptor seems to modify the activity of known agonists and antagonists at the P2Y11 receptor, as observed in the endocytosis experiments (section 3.3.1 and 3.3.2). BzATP was ineffective in inducing receptor-endocytosis and NF157 could not inhibit ATP-induced internalization of the P2Y11GFP receptor. Therefore, we also investigated the pharmacology of intracellular calcium rise induced by P2Y11GFP receptor activation in HEK293 cells. As described in methods, cells were subjected to single-cell calcium measurement applying the calcium indicator fura-2. Results are summarized in Fig. 31 and 32.
P2Y11GFP receptor-expressing HEK293 cells responded to the potent P2Y11 receptor agonist BzATP, but the recently developed P2Y11 receptor antagonist NF157 was not able to inhibit this response (Fig. 31A). However, when HEKP2Y11GFP cells were preincubated with the specific P2Y1 receptor antagonist MRS2179 (100 µM) no response to BzATP (100 µM) could be detected (Fig. 31A).
This antagonist sensitivity was different at the single P2Y11GFP receptor expressed in 1321N1 cells, where MRS2179 showed no effect on the activity of BzATP, but preincubation with NF157 totally abolished the activity of BzATP at the P2Y11 receptor (Fig. 31A). Thus, the interaction of the P2Y11GFP receptor with the endogenous P2Y1 receptor in HEK293 cells seems to influence the pharmacology of the receptor.
As the A268P2Y11GFP receptor did not functionally interact with the endogenous P2Y1
receptor in HEK293 cells with regard to internalization, we examined the influence of this mutant P2Y11 receptor on the pharmacological interaction with the P2Y1 receptor. Therefore, the intracellular calcium rise in HEKA268P2Y11GFP cells was measured. The cells responded to 100 µM ATP (Fig. 31B) in the same way as mock-transfected cells confirming the inability of 100 µM ATP to stimulate the P2Y11 receptor mutant. However, the more potent P2Y11
receptor agonist BzATP elicited a significant response at a concentration of 100 µ M at the R268A mutant (Fig. 31A) which was not seen in the mock-transfected cells (Fig. 31A).
Nevertheless, the amplitude of the response to 100 µM BzATP of the HEKA268P2Y11GFP cells was clearly smaller (25%) than the [Ca2+]i increase in HEKP2Y11GFP cells (Fig. 31A).
Pretreatment of the HEKA268P2Y11GFP cells with MRS2179 did not affect the action of BzATP at the P2Y11 receptor (Fig. 31A) in contrast to the observations made at the unmutated receptor (Fig. 31A). Thus, the R268A mutation of the P2Y11 receptor seems to disrupt the interaction with the P2Y1 receptor concerning the P2Y1 receptor mediated internalization of the P2Y11 receptor (section 3.3.3.1) and the ligand selectivities in inducing [Ca2+]i rise.
Moreover, real-time PCR showed that the differently transfected HEK293 cells display a comparable expression profile of P2Y receptor mRNAs, as depicted in the above figure at page 70 (Fig. 19). HEKA268P2Y11GFP, mock-transfected, as well as HEKP2Y11GFP cells all showed similar levels of P2Y1 receptor mRNA. The P2Y11 receptor expression was significantly increased in the accordingly transfected HEK293 cells compared to mock-transfected cells. The P2Y2 receptor expression was very weak and only clearly detectable in HEKP2Y11GFP cells. The P2Y6 receptor was not expressed in any of these cell clones. Thus, different P2Y receptor expression profiles apparently are not the reason for the diversity in the ligand selectivities at the unmutated and R268A mutated P2Y11 receptor in HEK293 cells.
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Figure 31: Peak values of intracellular calcium rise induced by P2Y receptor stimulation (A) Bar graphs display the [Ca2+]i rise in HEK293 cells transfected with GFP only (mock) (blank bar), P2Y11GFP (black bars), A268P2Y11GFP (striped bars) and the calcium rise in 1321N1P2Y11GFP cells (green bars). Cells were stimulated with BzATP at the indicated concentrations (µM). Preincubation with 100 µM MRS2179 or 1 µM NF157 is also indicated. [Ca2+]i rise was detected using fura-2 as described in methods.
(B) Bar graphs display the [Ca2+]i rise in response to 100 µM ATP of HEK293 cells transfected with GFP only (blank bar), P2Y11GFP (black bar), A268P2Y11GFP (striped bar). Data represent the mean±s.e.m obtained in at least three independent experiments analyzing 20-100 cells. The dashed lines in (A) and (B) give the comparison of the responses to that of mock transfected HEK293 cells.
Furthermore, a significant increase in intracellular calcium upon stimulation of the cells with 100 µM UDP was observed (Fig. 32). UDP is known to activate the P2Y6 receptor (Nicholas et al., 1996) but this receptor was not expressed in our HEK293 cells. Interestingly, only the green fluorescent, i.e. positively P2Y11GFP transfected, cells responded. After preincubation with MRS2179, the response was dramatically reduced (Fig. 32). The P2Y11
receptor antagonist NF157 was able to inhibit 46 % of the calcium response to 100 µM UDP.
When HEKA268P2Y11GFP cells were challenged with UDP, the intracellular calcium concentration was only slightly increased (Fig. 32), which was comparable to the value in HEKP2Y11GFP cells after incubation with MRS2179. Mock-transfected HEK293 cells (Fig.
32) and 1321N1P2Y11GFP cells (data not shown) showed no significant calcium response to UDP.
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Figure 32: Peak values of intracellular calcium rise induced by UDP in HEK293 cells
Bar graphs display the [Ca2+]i rise in HEK293 cells transfected with GFP only (blank bars), P2Y11GFP (black bars), A268P2Y11GFP (striped bars). Cells were stimulated with 100 µM UDP. Preincubation with 100 µM MRS2179 or 10 µM NF157 is indicated. [Ca2+]i rise was detected using fura-2 as described in methods.
Data represent the mean±s.e.m obtained in at least three independent experiments analyzing 40-100 cells.
3.3.6.2 Determination of cAMP accumulation
The P2Y11 receptor not only couples to the Gq protein, but also to the Gs protein, thereby inducing the activation of adenylyl cyclase and subsequent cAMP production in the cells.
Therefore, it was interesting to know whether the interaction of the P2Y11 receptor with the P2Y1 receptor also influences the receptor pharmacology with respect to cAMP accumulation.
HEKP2Y11GFP cells were stimulated with various agonists and/or antagonists, the reaction was stopped by addition of 0.1 M HCl and the cAMP content was measured after cell lysis, as explained in the methods section.
Stimulation of the cells with 100 µM BzATP led to a substantial increase in the cAMP concentration that was about 17-fold as compared to that of unstimulated cells (Fig. 33).
Preincubation with 100 µ M MRS2179 did not affect the response to BzATP. In contrast, cells preincubated with 10 µM NF157 showed a significantly reduced (by 40 %) response to BzATP. No cAMP accumulation was induced when cells were treated with 100 µM UDP or 10 µM 2-MeSADP. Thus, the pharmacology of adenylyl cyclase activation found in HEKP2Y11GFP cells represents the profile known for the P2Y11 receptor ligands.
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Figure 33: Stimulation of cAMP accumulation in HEKP2Y11GFP cells
Cells preincubated with IBMX (500 µM) and antagonists (as indicated) were stimulated with various nucleotides. The cAMP content was measured using an Enzyme-Linked-Immunoassay (EIA) as described in methods. Bar graphs represent the average stimulation (normalized to unstimulated cells) and s.e.m from at least three independent experiments. Asterisks indicate significance (* p<0.05) and ‘ns’ indicates non significant differences analyzed using one-side ANOVA and the Tukey’s test.