ATP evokes Ca2+ signals in cultured foetal human cortical astrocytes entirely through G protein‐coupled P2Y receptors

Abstract Extracellular ATP plays important roles in coordinating the activities of astrocytes and neurons, and aberrant signalling is associated with neurodegenerative diseases. In rodents, ATP stimulates opening of Ca2+‐permeable channels formed by P2X receptor subunits in the plasma membrane. It is widely assumed, but not verified, that P2X receptors also evoke Ca2+ signals in human astrocytes. Here, we directly assess this hypothesis. We showed that cultured foetal cortical human astrocytes express mRNA for several P2X receptor subunits (P2X4, P2X5, P2X6) and G protein‐coupled P2Y receptors (P2Y1, P2Y2, P2Y6, P2Y11). In these astrocytes, ATP stimulated Ca2+ release from intracellular stores through IP 3 receptors and store‐operated Ca2+ entry. These responses were entirely mediated by P2Y1 and P2Y2 receptors. Agonists of P2X receptors did not evoke Ca2+ signals, and nor did ATP when Ca2+ release from intracellular stores and store‐operated Ca2+ entry were inhibited. We conclude that ATP‐evoked Ca2+ signals in cultured human foetal astrocytes are entirely mediated by P2Y1 and P2Y2 receptors, with no contribution from P2X receptors.

Despite acceptance of the importance of ATP-evoked Ca 2+ signals in astrocytes, the evidence derives almost entirely from rodents, where mRNA for most P2 receptors has been detected, and both P2X and P2Y receptors have been implicated in Ca 2+ signalling (Fumagalli et al. 2003;Verkhratsky et al. 2009). However, there is a widespread assumption that most ATP-evoked Ca 2+ signals in rodent astrocytes are mediated by P2X 1/5 and P2X 7 receptors (Lalo et al. 2008(Lalo et al. , 2011. The evidence implicating P2X 7 receptors is controversial and derives largely from analyses of reactive astroctyes (Sim et al. 2004;Verkhratsky et al. 2009;Oliveira et al. 2011), where morphology and function are changed by the inflammatory mediators that are inevitably released during preparation of brain slices (Takano et al. 2014;Ben Haim et al. 2015). Furthermore, in humans, the P2X 5 subunit is truncated and retained within the endoplasmic reticulum (ER) (Kotnis et al. 2010). In cultured rodent astrocytes, P2Y 1 and P2Y 2 receptors, and to a lesser extent P2Y 4 receptors, can also initiate ATP-evoked Ca 2+ signals (Verkhratsky et al. 2009). Hence, even in rodent astrocytes, the identities of the receptors that mediate ATP-evoked Ca 2+ signals are unresolved (Fumagalli et al. 2003;Verkhratsky et al. 2009).
In human astrocytes, the receptors that mediate ATPevoked Ca 2+ signals are unknown. There has been no complete or quantitative analysis of mRNA expression levels for P2 receptors, although in cultures of human astrocytes mRNAs for P2Y 1 , P2Y 2 , P2Y 4 , P2X 4 , P2X 5 and P2X 7 receptors were detected (John et al. 2001;Narcisse et al. 2005;Hashioka et al. 2014). The only P2 receptor protein shown to be expressed is the P2X 7 subunit, but in healthy astrocytes it was exclusively expressed on intracellular membranes, and in brain sections it was detected only in diseased tissue (Narcisse et al. 2005). In the only analyses of Ca 2+ signals, 2-MeS-ATP and UTP evoked Ca 2+ signals in cultured human astrocytes, but the receptor pharmacology was not further defined (John et al. 1999). In another study, an agonist of P2X receptors (BzATP), which also stimulates P2Y 11 receptors (Communi et al. 1999), evoked a convincing Ca 2+ signal only in reactive astrocytes (Narcisse et al. 2005). Hence, the common but unverified, assumption that ATP-evoked Ca 2+ signals in healthy human astrocytes are largely mediated by P2X receptors requires further investigation (Burnstock 2008;Illes et al. 2012).
In this study, we define the receptors responsible for ATPevoked Ca 2+ signals in human astrocytes. We used cultures of foetal cortical human astrocytes to quantify mRNA expression for all P2 receptors, and we identified the P2 receptors through which ATP evokes Ca 2+ signals. There are limitations to the use of cultured cells, but for human brain tissue, it provides the only practicable means of directly measuring cytosolic Ca 2+ signals. Furthermore, it avoids the persistent astrogliosis caused by the traumatic injury and hypoxia inherent in preparing brain slices, which has been shown to affect expression of P2 receptors (Narcisse et al. 2005;Takano et al. 2014). Our results show that cultured human foetal astrocytes express mRNA for several P2X and P2Y receptors, but the Ca 2+ signals evoked by ATP are entirely mediated by P2Y 1 and P2Y 2 receptors.
Cell culture Human astrocytes isolated from foetal cortex were supplied as frozen cells that had not been passaged (catalogue number CC-2565, Lonza, Slough, UK). The cells were confirmed, by Lonza, to be free of infection with HIV-1 and hepatitis B and C, and we confirmed that they were free of mycoplasma. Astrocytes were grown at 37°C in humidified air containing 5% CO 2 , using astrocyte growth medium (Lonza) supplemented with 3% foetal bovine serum. Astrocyte growth medium includes human epidermal growth factor, insulin, ascorbic acid, gentamycin and L-glutamine. Cells were passaged using trypsin, according to the supplier's instructions, when they reached 70-80% confluence. Cells were used for up to four passages after receipt, during which they maintained an astrocyte-like morphology and expressed glial fibrillary acidic protein, assessed by qPCR and immunostaining.

Quantitative PCR
For quantitative PCR (qPCR), confluent cultures of astrocytes in 24well plates were lysed (200 lL cell processing buffer/well), mRNA was then isolated from the lysate (4 lL) and cDNA was synthesized using Fastlane cell cDNA kit (Qiagen,Crawley, UK). The cDNA was diluted fivefold with RNAase-free water. Incubations for qPCR included Rotor-Gene SYBR TM Green PCR master mix (10 lL), cDNA (5 lL), Quantitect primer assay (2 lL, Table S2) and RNAase-free water (3 lL). In negative controls, the primers were omitted during qPCR or the reverse-transcriptase was omitted during cDNA synthesis. A Rotor-Gene 6000 thermocycler (Qiagen) was used for qPCR with a denaturation step (95°C, 5 min), 40 amplification cycles (5 s at 95°C, 10 s at 60°C) and then a melting curve (70-95°C). Expression of mRNA relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was calculated from: Rela- , where E is the amplification efficiency, calculated as 10 m , where m is the average increase in fluorescence for four cycles after the cycle threshold C T for the indicated PCR product. The effectiveness of all primer pairs was verified using BioBank generic pooled cDNA (Primerdesign Ltd, Chandler's Ford UK). All primers included in this study amplified a single product from the BioBank pooled cDNA ( Figure S1). The melting temperatures of all products amplified from cDNA from astrocytes were identical to the respective BioBank controls. Results are reported as means from cDNA samples independently obtained from three different cell cultures.
Fluorescence (excitation at 490 nm, emission at 520 nm) was recorded at 1.44-s intervals using a FlexStation III fluorescence platereader (MDS Analytical Technologies, Wokingham, UK), which allows automated fluid additions during the recording (Tovey et al. 2006). Fluorescence (F) was calibrated to intracellular free Ca 2+ concentration ([Ca 2+ ] i ) from: ½Ca 2þ i ¼ K d FÀFmin FmaxÀF , where K D is the equilibrium dissociation constant of fluo-8 for Ca 2+ (389 nM), F min and F max are the minimal and maximal fluorescence values determined after addition of Triton X-100 (0.2%) in Ca 2+ -free HBS with BAPTA (10 mM, F min ) or ionomycin (10 lM) in normal HBS (F max ).

Measurement of [Ca 2+ ] i in single astrocytes
Almost confluent cultures of astrocytes grown on fibronectin-coated eight-well imaging slides (Thistle Scientific Ltd, Glasgow, UK) were loaded with fura-2 by incubation with fura-2 AM (2 lM) in HBS containing 2.5 mM probenecid (45 min, 20°C). After a further 45 min in the same medium without fura-2 AM, the cells were used for experiments at 20°C in HBS without probenecid. Imaging was performed using an Olympus IX71 inverted fluorescence microscope with alternating excitation (340 nm and 380 nm) provided by a Xe-arc lamp at 1-s intervals. Emission was recorded at 510 nm using a Luca EMCCD camera (Andor Technology, Belfast, UK) and MetaFluor software (Molecular Devices, Sunnyvale, CA, USA). Background-corrected ratios of F 340 /F 380 fluorescence were used to determine whether ligands evoked increases in [Ca 2+ ] i .

Statistical analysis
For each experiment, the concentration-response relationship was fitted to a logistic equation (GraphPad Prism 5, La Jolla, CA, USA) from which the maximal amplitude and pEC 50 values (Àlog of the half-maximally effective drug concentration) were determined. All analyses, except when otherwise stated, show pooled results from cells from two donors (Lonza lot numbers: 0000289765 and 0000402839). Key results from these and a third donor (0000514417) are shown individually in Figure S2. Results are presented as means AE SEM of values from at least three independent experiments. Sample sizes (n) refer to independent experiments.

Results
ATP evokes Ca 2+ release and Ca 2+ entry in cultured human foetal astrocytes ATP evoked a concentration-dependent increase in [Ca 2+ ] i (pEC 50 = 5.94 AE 0.03, n = 8) in populations of cultured foetal human cortical astrocytes. Similar results and with similar sensitivities to ATP, but with Ca 2+ signals of different amplitude, were observed in cells from three different donors ( Figure S2A). Removal of extracellular Ca 2+ affected neither the peak amplitude of the increase in [Ca 2+ ] i nor its sensitivity to ATP (pEC 50 = 5.64 AE 0.08, n = 5), but the sustained phase of the response was abolished ( Fig. 1a and b). These results establish that release of Ca 2+ from intracellular stores and Ca 2+ entry across the plasma membrane contribute to the ATP-evoked Ca 2+ signals. U73122, an inhibitor of phospholipase C (Bleasdale et al. 1990), but not its inactive analogue (U73343), caused a concentrationdependent inhibition of the ATP-evoked Ca 2+ signals (Fig. 1c). 2-APB, an antagonist at IP 3 Rs (Saleem et al. 2014), also inhibited ATP-evoked Ca 2+ signals (Fig. 1d). Neither 2-APB nor U73122 completely blocked the response to ATP, but the range of useable concentrations is limited by off-target effects of the inhibitors (Grierson and Meldolesi 1995;Mogami et al. 1997;Peppiatt et al. 2003). These results demonstrate that ATP-evoked Ca 2+ signals are at least substantially dependent on stimulation of PLC and IP 3evoked release of Ca 2+ from intracellular stores.
We used qPCR with primers demonstrated to selectively amplify mRNA encoding each of the human P2Y and P2X receptors (Table S2) to quantify expression of these mRNAs in cultured foetal human cortical astrocytes. The results confirmed expression of mRNA for four of the eight subtypes of P2Y receptors (P2Y 1 , P2Y 2 , P2Y 6 and P2Y 11 ) and three of the seven subunits of P2X receptors (P2X 4 , P2X 5 and P2X 6 ) (Fig. 1e). There was no detectable expression of mRNA for the remaining P2Y or P2X receptors, despite the proven effectiveness of the primers used ( Figure S1).

P2X receptors do not evoke Ca 2+ signals
Since some P2Y receptors, but no P2X receptors, can stimulate PLC (Burnstock and Kennedy 2011), our results so far suggest a major (and perhaps exclusive) role for P2Y receptors in initiating ATP-evoked Ca 2+ signals in human astrocytes. This contrasts with the prominent role ascribed to P2X receptors in rodent astrocytes. We therefore assessed whether P2X receptors contribute to the Ca 2+ signals evoked by ATP in human astrocytes.

Results
(means AE SEM from three independent samples, each measured in duplicate) are expressed as percentages of all P2 receptor mRNA. There was no detectable expression of mRNA for the remaining P2Y (4, 12-14) or P2X (1-3, 7) receptor subtypes, although the primers used were all shown to be effective ( Figure S1).  In these experiments, the peak increases in [Ca 2+ ] i (D[Ca 2+ ] i ) evoked by a,b-meATP and BzATP were 5 AE 0 nM and 5 AE 3 nM respectively (n = 3); the parallel measurements of ATP-evoked D[Ca 2+ ] i were 132 AE 26 nM and 150 AE 16 nM. We avoided higher concentrations of a,b-meATP (100 lM) and BzATP (300 lM) because they evoked Ca 2+ signals in Ca 2+ -free HBS (not shown).
We next attempted to eliminate the Ca 2+ signals evoked by P2Y receptors to unmask any possible contribution from P2X receptors. This required inhibition of both the Ca 2+ release and Ca 2+ entry components of the response evoked by P2Y receptors (Fig. 1a and b). Thapsigargin, which inhibits Ca 2+ pumps in the ER, is commonly used to deplete the ER of Ca 2+ and to thereby stimulate store-operated Ca 2+ entry (SOCE) (Parekh and Putney 2005). We confirmed that thapsigargin stimulated SOCE in human astrocytes (Fig. 2c). Pre-treatment of astrocytes with three structurally unrelated inhibitors of SOCE, BTP-2 (10 lM), SKF96365 (10 lM) and 2-APB (100 lM) (Bootman et al. 2002;Liou et al. 2005;Ohga et al. 2008) almost abolished the SOCE evoked by thapsigargin (Fig. 2d). Although 2-APB inhibits both IP 3 R and SOCE, its effects on thapsigargin-evoked Ca 2+ entry are probably due to it inhibiting formation of the STIM1 puncta that stimulate SOCE (DeHaven et al. 2008).
In astrocytes pre-treated with thapsigargin to deplete intracellular Ca 2+ stores and so prevent IP 3 -evoked Ca 2+ release, and with BTP-2, SKF96365 or 2-APB present to inhibit SOCE, a normally maximally effective concentration of ATP (100 lM) had no significant effect on [Ca 2+ ] i ( Fig. 2e and f). Similar results were observed in cells from all three donors ( Figure S2B). These results confirm that the Ca 2+ entry evoked by ATP is likely mediated by SOCE, and that there is no additional response to ATP mediated by P2X receptors.
To exclude any possible off-target effects of the SOCE inhibitors on P2X receptors, we compared the effects of ATP in HBS on astrocytes with and without prior thapsigargin treatment. This experiment is practicable because the amplitude of the Ca 2+ signal evoked by SOCE decays relatively quickly in the continued presence of extracellular Ca 2+ (Fig. 2c), such that the small residual SOCE-mediated Ca 2+ signal detected after 15 min would not obscure a response to ATP. Under these conditions, addition of ATP (100 lM or 1 mM) to thapsigargin-treated cells in normal HBS had no significant effect on [Ca 2+ ] i ( Fig. 2g and h). The lack of response to such high concentrations of ATP excludes a role for P2X receptors, including P2X 7 receptors which have low affinity for ATP (Surprenant et al. 1996). These results demonstrate that P2X receptors make no detectable contribution to the Ca 2+ signals evoked by ATP in cultured human cortical astrocytes, despite evidence that the cells express mRNA for three P2X receptor subunits (Fig. 1e).
An increase in [Ca 2+ ] i has been reported to stimulate translocation of P2X 4 receptors from intracellular membranes to the plasma membrane (Qureshi et al. 2007;Vacca et al. 2009). We therefore considered whether release of Ca 2+ from intracellular stores might stimulate a similar translocation of P2X receptors in human astrocytes and thereby allow ATP to sequentially activate P2Y and then P2X receptors. However, when astrocytes were first stimulated with ADP to activate P2Y (but not P2X) receptors, there was the expected increase in [Ca 2+ ] i , but subsequent addition of a,b-meATP to stimulate P2X receptors (30 lM after 5 min) evoked no further increase in [Ca 2+ ] i ( Figure S3).
Collectively, these results demonstrate that the Ca 2+ signals evoked by ATP in cultured human cortical astrocytes are entirely mediated by P2Y receptors with no detectable contribution from P2X receptors.
P2Y 1 and P2Y 2 receptors mediate ATP-evoked Ca 2+ signals All four of the P2Y receptor subtypes for which mRNA was detected in human astrocytes (P2Y 1 , P2Y 2 , P2Y 6 and P2Y 11 ) are coupled to G q/11 and can thereby stimulate PLC. We used ligands that distinguish between the subtypes for which mRNA was detected to resolve the contributions of different P2Y receptors to the ATP-evoked Ca 2+ signals (Table S1).
To determine whether activation of P2Y 1 receptors (with ADP) and of P2Y 2 receptors (with UTP) are entirely responsible for the Ca 2+ signals evoked by ATP, we compared the maximal amplitudes of the responses evoked by the three stimuli in parallel measurements. The D[Ca 2+ ] i evoked by ATP, ADP and UTP were 142 AE 5 nM, 88 AE 11 nM and 51 AE 13 nM (n = 3) respectively. Hence, the sum of the responses to ADP and UTP (139 AE 25 nM) was not significantly different from the response evoked by ATP (142 AE 5 nM). These results confirm that ATP evokes Ca 2+ signals through P2Y 1 and P2Y 2 receptors, but they do not resolve whether the two receptors are expressed in different cells or whether both contribute to the responses in individual cells. We therefore examined the responses of single fura-2-loaded cells to ATP, UTP and ADP.
In these single-cell analyses, 87 AE 6% of cells responded to ATP (101 cells, 4 independent fields), 65 AE 5% responded to ADP (502 cells, 13 fields) and 41 AE 6% responded to UTP (386 cells, 11 fields), suggesting that at least 22% of ATP-responsive cells express both P2Y 1 and P2Y 2 receptors. Analyses of responses to sequential stimulation with ADP and UTP revealed that 59 AE 8% of the cells that responded to ADP then responded to UTP (204 cells, 7 fields), while 92 AE 6% of cells that responded to UTP responded to a subsequent challenge with ADP (152 cells, 5 fields), suggesting that about half of the cells responded to both stimuli. These results demonstrate that most cells respond to ATP and that many express both P2Y 1 and P2Y 2 receptors. We considered whether autocrine release of ATP might contribute to the sustained phase of the Ca 2+ signal evoked by selective activation of P2Y receptors. This seems unlikely, since in cell populations the relative amplitudes of the initial and sustained phases were similar for cells stimulated with ATP to activate all P2Y receptors or with ADP to activate only P2Y 1 receptors ( Fig. 1a and S3). Furthermore, in our single-cell analyses, none of the cells that failed to respond initially to selective activation of P2Y 1 receptors (176 cells) or P2Y 2 receptors (228 cells) responded during the next 3-6 min with a detectable increase in [Ca 2+ ] i .

Discussion
We provide the first complete quantitative analysis of mRNA expression for P2 receptors in cultured foetal human cortical astrocytes, and a comprehensive pharmacological characterization of ATP-evoked Ca 2+ signals. We showed that mRNAs for four P2Y receptors (P2Y 6~P 2Y 11 > P2Y 2 > P2Y 1 ) and three P2X receptor subunits (P2X 6 > P2X 4 > P2X 5 ) are expressed. There was no detectable mRNA for any of the remaining P2 receptors (Fig. 1e). The expression pattern is broadly consistent with previous studies of cultured human astrocytes from both adult (Hashioka et al. 2014) and foetal tissue (John et al. 2001;Narcisse et al. 2005), which examined mRNA for only seven of the fifteen P2 receptors, and detected mRNA for P2Y 1 , P2Y 2 , P2Y 4 , P2X 4 , P2X 5 and P2X 7 receptors. The notable differences are the absence of mRNA for P2Y 4 and P2X 7 receptors in our analyses, with the latter perhaps explained by the presence of fewer reactive astrocytes in our analysis (Narcisse et al. 2005). Neither we nor others have verified the relationship between mRNA and protein expression in human astrocytes because the P2 receptor-selective antibodies generally lack specificity (Sim et al. 2004;Takano et al. 2014).
In keeping with many analyses of rodent astrocytes, ATP evoked an increase in [Ca 2+ ] i in both confluent populations of human cultured foetal astrocytes and sub-confluent single cells (Verkhratsky et al. 2009). In human astrocytes, the initial response to ATP was because of Ca 2+ release from intracellular stores through IP 3 receptors ( Fig. 1a-d), but the sustained response required Ca 2+ entry across the plasma membrane. The Ca 2+ entry had pharmacological properties typical of SOCE ( Fig. 2e and f). In most cells, receptors that stimulate PLC usually activate SOCE (Parekh and Putney 2005), and in rodent astrocytes P2Y receptors have been shown to evoke Ca 2+ entry by stimulating PLC (Fumagalli et al. 2003), but SOCE evoked by P2Y receptors has not, to the best of our knowledge, been previously reported for human astrocytes. These results are not consistent with the prominent role ascribed to P2X receptors in rodent astrocytes. Since mRNAs for three P2X receptor subunits were expressed in human astrocytes, we looked more closely to determine whether there was any underlying contribution from P2X receptors to ATP-evoked Ca 2+ signals. ATP analogues that would be expected to stimulate human P2X receptors assembled from P2X 4 , P2X 5 or P2X 6 subunits (a,b-meATP and BzATP) did not increase [Ca 2+ ] i ( Fig. 2a and b). Furthermore, under conditions where responses from IP 3 receptors and SOCE were inhibited, there was no response to ATP ( Fig. 2e and f). We confirmed that this lack of effect of ATP was not due to off-target effects of the inhibitors used to block SOCE ( Fig. 2g and h). Hence, whether assessed using ATP analogues selective for P2X receptors or ATP itself, there is no evidence that P2X receptors evoke Ca 2+ signals in cultured human foetal astrocytes. Finally, we considered whether the IP 3 -evoked Ca 2+ signal might stimulate translocation of intracellular P2X 4 receptors to the plasma membrane (Qureshi et al. 2007;Vacca et al. 2009), but we found no evidence to suggest that Ca 2+ release and SOCE unmasked a response to P2X receptors ( Figure S3).
The only published argument suggesting a role for P2X receptors in Ca 2+ signalling in normal human astrocytes derives from their expression of mRNA for some P2X receptor subunits (John et al. 2001;Narcisse et al. 2005;Hashioka et al. 2014). Our results demonstrate that although cultured foetal cortical human astrocytes express mRNA for some P2X receptor subunits (Fig. 1e), P2X receptors do not contribute to the Ca 2+ signals evoked by ATP (Fig. 2). Instead, we have shown that two of the four P2Y receptor subtypes for which mRNA was detected, P2Y 1 and P2Y 2 receptors, are entirely responsible for ATP-evoked Ca 2+ signals ( Fig. 3 and Figure S2). Our conclusion is consistent with a previous report in which two non-selective analogues, UTP and 2-MeS-ATP, which would together activate P2Y 1 and P2Y 2 receptors, evoked Ca 2+ signals in human astrocytes (John et al. 1999).
Our analyses of mRNA for P2X receptors were not predictive for expression of functional plasma membrane receptors. Others have also noted expression of mRNA for P2 receptors for which there was no corresponding functional response (Fumagalli et al. 2003). For P2X 5 subunits, a likely explanation is that the human protein is truncated and retained in the ER, where it may also trap other P2X subunits with which it can oligomerize (P2X 4 and P2X 6 ) (Torres et al. 1999;Kotnis et al. 2010). For P2Y receptors too, the most abundant mRNAs (for P2Y 6 and P2Y 11 ) were not associated with expression of functional P2Y receptors. In rodents too, there is no functional response to P2Y 6 receptors, although their mRNA is expressed (Fumagalli et al. 2003). These observations are relevant because mRNA expression in astrocytes has often been used to infer the likely identity of the receptors that mediate ATP-evoked Ca 2+ signals (Verkhratsky et al. 2009).
We conclude that in cultured foetal cortical human astrocytes, ATP evokes Ca 2+ signals that are entirely mediated by P2Y 1 and P2Y 2 receptors, each of which stimulates PLC and thereby IP 3 -evoked Ca 2+ release and SOCE. Many astrocytes express both of these receptors, but some express only one or the other. We have not further explored this heterogeneity. Although mRNA for P2X receptor subunits is expressed, P2X receptors do not contribute to ATP-evoked Ca 2+ signals. Figure S2. ATP evokes Ca 2+ signals through P2Y receptors in astrocytes from three donors. Figure S3. Stimulation of P2Y receptors does not cause translocation of functional P2X receptors to the plasma membrane. Figure S4. Neither P2Y 6 nor P2Y 11 receptors evoke Ca 2+ signals in cultured human foetal astrocytes. Figure S5. 2 0 -azido-UTP does not evoke Ca 2+ signals. Figure S6. Effects of MRS2179, a selective antagonist of P2Y 1 receptors, on the Ca 2+ signals evoked by ATP and MRS2365. Table S1. Properties of the drugs used. Table S2. Primers used for qPCR analyses. Table S3. Ca 2+ signals evoked by P2Y-selective agonists in cultured human foetal astrocytes.