ATP and Its Metabolite Adenosine Act Synergistically to Mobilize Intracellular Calcium via the Formation of Inositol 1,4,5-Trisphosphate in a Smooth Muscle Cell Line*

on the of 1,4,5-trisphosphate


ATP and Its Metabolite Adenosine Act Synergistically to Mobilize Intracellular Calcium via the Formation of Inositol 1,4,5-Trisphosphate in a Smooth Muscle Cell Line*
Par GerwinsS and Bertil B. Fredholm From the Department of Pharmacology, Karolinska Institutet, Box 60 400, S-104 01 Stockholm, Sweden Interactions between ATP and adenosine on the formation of inositol 1,4,5-trisphosphate (Ins (1,4,5)P3) and mobilization of intracellular calcium were investigated in the smooth muscle cell line DDTl MF-2. Activation of adenosine Al receptors with adenosine or cyclopentyladenosine (CPA) or of nucleotide receptors with ATP increased both Ins (1,4,5)P3 formation and intracellular calcium concentrations.
ATP (ECso 4 p~) and CPA (ECso 4 nM) both increased intracellular calcium levels. The effect of ATP was partially sensitive to PTX treatment, whereas the effect of CPA was blocked both by PTX and by DPCPX. Concentrations of ATP and CPA that by themselves were insufficient to raise intracellular calcium were able to do so when combined. The synergy between ATP and CPA on the mobilization of intracellular calcium was abolished after treatment of cells with PTX or when DPCPX was included in the experiment.
Since ATP was metabolized by ecto-enzymes to ADP, AMP, and adenosine, we also examined whether adenosine formed from ATP could enhance the ATP effects on Ins(l,4,6)P3 accumulation. Indeed, the addition of the A1 receptor antagonist DPCPX or removal of endogenous adenosine by inclusion of adenosine deaminase in the experimental medium significantly attenuated the ATP response, and the two treatments did not have additive effects.
The present study thus demonstrates that in a clonal cell line two types of receptors increase phospholipase C activity, but via different pathways; nucleotide receptors appeared to act via partially PTX-insensitive, and A1 receptors via PTX-sensitive G-proteins. ATP and CPA are not only able per se to induce for-* These studies were supported by Swedish Medical Research Council Grant K92-04P-09717-02 (Project 2553), the Petrus and Augusta Hedlunds Foundation, the Swedish Association for Medical Research, and Karolinska Institutet. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ATP is not only a key intracellular energy donor but it also has extracellular signaling functions and acts like a transmittor (1). It acts on specific receptors (P2 receptors) that are tentatively divided into four subtypes, P2x, P2u, P2z, and P2T (1,2). Among these, the PzU receptor, probably via G-proteins,' stimulates phospholipase C (3)(4)(5). In addition a socalled nucleotide receptor that is activated equally well by UTP and ATP and is linked to phospholipase C has been postulated (6).
Activation of phospholipase C (7) results in the formation of two second messengers: Ins (1,4,5)P3, which mobilizes calcium from intracellular stores (8), and diacylglycerol, which activates protein kinase C (9). Extracellular ATP is rapidly degraded to ADP, AMP, and adenosine (10). One of the metabolites, adenosine, is a ubiquitous modulator in its own right and acts via specific adenosine receptors. These are classified into subtypes, A, and A2, originally depending on whether they stimulate (A2) or inhibit (A1) adenylylcyclase (11,12) and later depending on the order of potency of agonists and antagonists in receptor binding studies (13). Adenosine AI receptors not only mediate inhibition of adenylylcyclase but are also linked to potassium and calcium channels (14,15).

A T P and
Adenosine Interactions on Phospholipase C is evidence that in some cells activation of adenosine receptors can directly stimulate phospholipase C (27-30). However, the receptor involved in these effects was not typical for either AI or AB receptors, and it has even been proposed that a novel adenosine receptor is involved (30, 31). Since it is known that ATP is degraded to adenosine and that adenosine via adenosine receptors can modify the formation of inositol phosphates via several receptors, we wanted to directly investigate whether adenosine formed from ATP could, via A1 receptors, influence the ability of ATP, acting on nucleotide receptors, to form Ins(1,4,5)P3 and elevate intracellular calcium. Another purpose of the present study was to investigate if adenosine A1 receptors are able to couple to more than one intracellular signal transduction pathway. We have used a smooth muscle cell line, DDT, MF-2 (32), that is known to express AI receptors negatively coupled to adenylylcyclase (33, 34) and some type of receptor for ATP that is coupled to phospholipase C (35).
Cell Culture-DDT, MF-2 smooth muscle cells, originally isolated from a steroid-induced leiomyosarcoma of Syrian hamster vas deferens (32), were obtained from the American Type Culture Collection (ATCC). Cells were grown in suspension, maintained in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g of glucose/liter (containing 5% fetal calf serum), 100 units/ml penicillin, 100 pg/ml streptomycin, and 2 mM L-glutamine, at 37 "C in 5% C02, 95% air. Cells were subcultured three times weekly and used at density of approximately lo5 cells/ml. Cell viability was more than 90% as assessed by the exclusion of trypan blue.
Determination of In.s(l,4,5)P3-Cells were washed once in assay medium (DMEM buffered with 20 mM HEPES, pH 7.4) and resuspended in assay medium to a concentration of 3 X lo6 cells/ml. Aliquots (0.2 ml = 6 X lo5 cells) were transferred to test tubes and preincubated for 20 min at 37 'C in a water bath before addition of indicated drugs (0.1 ml). Lithium was omitted to avoid any interaction with the formation of inositol phosphates. Reactions were terminated by the addition of perchloric acid to a final concentration of 0.4 M and the tubes placed on ice for 1 h. Samples were neutralized with 4 M KOH, 1 M Tris, 60 mM EDTA and frozen until analyzed.
CAMP assay-After being washed once with assay medium, cells were resuspended in the same medium to a density of 1.4 X lo5 cells/ ml. Aliquots (0.35 ml = 0.5 X lo5 cells) were transferred to test tubes and the indicated drugs added together with 30 pM of the phosphodiesterase inhibitor rolipram to a final volume of 0.5 ml. Reactions were terminated after incubation at 37 "C by the addition of perchloric acid to a final concentration of 0.4 M. Samples were neutralized with 5 M KOH, 1 M Tris and the cAMP content in the supernatants determined with a protein binding assay (37), where bound [3H] cAMP was separated from free by rapid filtration over glass fiber filters (Skatron AS).
Measurement of Intracellular Concentrations of Free Calcium-Cells were washed and resuspended in Hank's balanced salt solution (HBSS, 1.2 mM CaC12 supplemented with 0.1% bovine serum albumin and 20 mM HEPES, pH 7.4) to a concentration of lo6 cells/ml and loaded with 5 p~ of Fura 2-AM for 40 min at 37 "C. After the loading period cells were washed twice in HBSS and resuspended to a concentration of IO6 cells/ml. Prior to the measurements, cells were washed once more and then placed in a cuvette (IO6 cells in ' 2 ml of HBSS) and the intracellular calcium concentration determined at 30 "C in a dual-wavelength Sigma ZFP22 fluorometer by using the ratio of excitation wavelengths 334/366 nm with emission cut off at 500 nm. Free calcium concentration was calculated as previously described (38).
Analysis of Adenine Nucleotides-After one wash, cells were resuspended in HBSS at a concentration of 2.5 x lo6 cells/ml. Aliquots (0.4 ml = lo6 cells) were transferred to test tubes and ATP (0.1 ml) added to a final concentration of M. Incubations were terminated by the addition of perchloric acid, 0.4 M final concentration. After neutralization with 4 M KOH the amount of adenine nucleotides was determined in the supernatant by reversed-phase HPLC using a 15cm Nucleosil5 C,, column. The identities of adenine nucleotides were ascertained by "spiking" of samples with authentic nucleotides and in the case of adenosine by treatment with adenosine deaminase. In experiments where we determined the amount of adenosine released upon stimulation of cells with ATP, cells were prelabeled with [3H] adenine (5 pCi/ml, 1 h) and activated as described above with the exception that reactions were terminated by rapid centrifugation and aspiration of the supernatants, which were immediately mixed with perchloric acid (0.4 M ) and neutralized with KOH prior to HPLC analysis. The amount of released [3H]adenosine was analyzed by measuring the radioactivity in fractions collected after HPLC separation.
Data Ana1ysb"ose-response curves were generated by using the GraphPad (IS1 Software) program. Statistical comparisons between different drug treatments were made using analysis of variance using the Statgraphics (Statistical Graphics Corp.) program with a confidence level of 95% or using Student's t test (GraphPad InStat; IS1 Software). Data are presented as mean & S.E.

ATP and
Adenosine Interactions on Phospholipase C 16083 base-line values within 10 min (Fig. 2 ) . Adenosine (10 p~) and the selective adenosine A1 receptor agonist CPA (100 nM) also increased intracellular levels of Ins(1,4,5)P3 (Fig. 2 ) . The peak was reached after approximately 1 min, and control values were obtained after 5 min (Fig. 2 ) . Furthermore, when combined, ATP and CPA acted in synergy between 0.5 and 3 min after addition of the two drugs ( p < 0.05, Student's t test; n = 4), and the effect of ATP was reduced by the addition of the A1-specific antagonist DPCPX (Fig. 2 ) .
CPA appears to act via typical AI receptors since the EC50 value was 10 f 1 nM (n = 3; Fig. 3). Treatment of cells with pertussis toxin (200 ng/ml, 4 h), which completely prevents subsequent ADP-ribosylation of purified membranes by activated PTX (33), abolished the response to CPA (Fig. 3). The effect of an inactive dose of ATP (1 p~) was not altered by CPA over the concentration range studied, but the effect of a high concentration of ATP (0.1 mM) was enhanced by CPA in a concentration-dependent manner with an EC5a of 3 f 0.7 nM ( n = 3; Fig. 3). This enhancement was blocked by the AI receptor antagonist DPCPX with a Ki of 0.8 nM (Fig. 4), further supporting the idea that the effects are mediated with A, receptors.
DPCPX did not have any effects on its own on intracellular levels of Ins(1,4,5)P3 (data not shown) but significantly ( p < 0.05, analysis of variance) attenuated ATP-induced Ins(1,4,5)P3 formation (Fig. 2 ) . The inclusion of adenosine deaminase (preincubation of cells for 1 h at 37 "C with 3 IU/ ml and a maintained concentration of 3 IU/ml during the experiments) by itself reduced the peak Ins ( formation (Fig. 6). Thus, these data indicate that adenosine, possibly formed from ATP via degradation by ecto-enzymes, acts in synergy with ATP on Ins(1,4,5)P3 formation. Consequently the enhancement of the ATP response by simultaneous activation of adenosine AI receptors must be assessed by comparing the responseATp+cpA with the responsecpA + responseATp+Dpcpx, where DPCPX is added to antagonize activation of A, receptors by endogenous ligand. These comparisons, which are shown in Figs. 2-5, demonstrate that the synergistic interaction is highly significant. Degradation of ATP-In view of the possible formation of adenosine from the added ATP we examined the degradation of ATP by DDT, MF-2 cells. Incubation of cells with ATP showed a time-dependent metabolism of ATP to form ADP, AMP, and adenosine (Fig. 7). Inclusion of adenosine deaminase in the experiments completely prevented the formation of adenosine from ATP (data not shown).
[3H]Adenine was incorporated into cells that were then stimulated with ATP (0.1 mM) for 0-60 min, and the resulting amount of [3H]adenosine in the media was determined by HPLC analysis. The results showed that there was no release of adenosine from the cells when stimulated with ATP (data not shown).
Specificity of the AdenosinelATP Interaction on Ins(1,4,5)P3 Formation and Possible Mechanisms-In DDT, MF-2 cells Adenosine as an impurity in the ATP solution contributed less than 5 nM to the time zero adenosine levels in all experiments, a concentration that has no effect on Ins(1,4,5)P3 formation (data not shown). The cells themselves contributed 10 nM or more. These values are subtracted to show only the adenosine increase due to ATP breakdown. Inclusion of adenosine deaminase in the experimental media completely prevented the formation of adenosine.
Ins(1,4,5)P3 formation is increased not only by ATP, but also by bradykinin, histamine, and phenylephrine (acting on 01,adrenoceptors) (results not shown). The effects of bradykinin and histamine were enhanced by adenosine, but there was no clear enhancement of the rather weak effect of phenylephrine. Furthermore, adenosine and adenosine analogues were alone in enhancing the actions of ATP (not shown). This is interesting because the response to the adenosine analogues is the only one that is completely eliminated by treating the cells with pertussis toxin (not shown). Adenosine A, receptors are coupled to adenylylcyclase in a negative manner via pertussis toxin-sensitive inhibitory Gproteins, presumably Gi2 o r Gi3, in DDT, MF-2 cells (39). In DDT, MF-2 cells CPA inhibited isoprenaline-stimulated cAMP accumulation with an ECso of 1.9 f 0.1 nM ( n = 3; data not shown). To exclude the possibility that the synergy between CPA and ATP on Ins(1,4,5)P3 formation was due to inhibition of cAMP we performed experiments where cAMP was increased with forskolin (forskolin increased intracellular cAMP levels from 27 k 2 to 149 f 24 pmol/106 cells, n = 4). Forskolin treatment reduced the ATP-induced Ins( 1,4,5)P3 accumulation, but did not affect the synergy between ATP and CPA (Fig. 8). Furthermore, ATP, CPA, adenosine, and DPCPX did not significantly affect basal cAMP levels when added alone or in combination (data not shown).
In many cells adenosine A, receptors activate potassium channels, but the response to the combination of CPA and ATP was not influenced by two potassium channel antagonists, 4-aminopyridine (0.3 mM) or tetraethylammonium (5 mM), alone or in combination (not shown). Indomethacin (10 p~) and dexamethasone (1 p~) alone or in combination were similarly ineffective (not shown). In order to examine the possible involvement of protein kinase C, cells were incubated with PMA (250 nM) 18 h before the experiment to downregulate protein kinase C. PMA treatment tended to reduce the ATP response in itself, but did not eliminate the synergistic response to CPA and ATP. PMA (250 nM) added at the same time as ATP o r CPA was not able to substitute for one or the other of these agonists (not shown). Thus, there is no evidence that CAMP, membrane potential, activation of phospholipase A2, or protein kinase C is involved in the interaction between ATP and adenosine analogues. Mobilization of Intracellular Calcium-ATP and CPA independently increased intracellular free calcium with ECso values of 4 f 0.9 p M and 4 f 0.9 nM, respectively ( n = 4; Fig.  9). After an initial peak, a sustained plateau phase followed (Fig. 10, A and B ) . The plateau phase was eliminated if extracellular calcium was removed by the addition of 3 mM EGTA (Fig. 10, A and B ) . The ATP response was partially sensitive to treatment of cells with pertussis toxin (200 ng/ ml, 4 h) with no significant change in the dose required for half-maximal response (8 -+ 2 p~ in treated cells compared to 4 k 0.9 p M in control cells, p > 0.05, Student's t test; n = 3; Figs. 9 and lOC). On the other hand, CPA-mediated calcium increase was completely abolished after pertussis toxin treatment (Figs. 9 and 1OC). CPA (10 nM) induced calcium mobilization was blocked by the selective AI receptor antagonist DPCPX (100 nM; Fig. lOD), whereas ATP-induced calcium increase was unaffected by DPCPX (data not shown). Thus, in contrast to the situation with Ins (1,4,5)P3 there is no evidence that endogenous adenosine augments the ATP response on calcium mobilization.
Low doses of ATP (0.3 p~) and CPA (0.3 nM), which were unable to raise intracellular calcium on their own, could when combined cause a clearcut increase in intracellular calcium (Fig. 10E).

ATP and
Adenosine Interactions on Phospholipase C When we combined submaximal doses of ATP (1 FM) and CPA (1.8 nM) they acted in concert and gave a recording with a distinctive initial peak, something that was not seen when they were added alone (Fig. 10F).

DISCUSSION
We have confirmed and extended previous findings that ATP increases Ins (1,4,5)P3 and intracellular calcium in DDTl MF-2 cells (35). The EC6,,values (21 and 4 PM) were in agreement with studies in other types of cells (40-45) as well as in DDT, MF-2 cells (35). The receptor involved is best described as a nucleotide receptor rather than as a Ppy receptor (6) since the potency ratio was UTP = ATP > ADP >> 2methylthio-ATP. The finding that intracellular calcium was increased at lower concentrations of ATP than was Ins (1,4,5)P3 indicates that the calcium response is fully activated even at a submaximal increase in Ins (1,4,5)P3, in agreement with previous reports (35,41). The time course for Ins (1,4,5)P3 formation is consistent with results from other cells (42, 44), but differs from an earlier study on DDT, MF-2 cells where a second increase in Ins ( 1,4,5)P3 was seen after 5 min (35). A possible explanation for this discrepancy is that we measured the endogenously formed Ins (1,4,5)P3, whereas in the earlier study Hoiting et al. measured the radioactively labeled Ins (1,4,5)P3.
ATP caused a rapid rise in intracellular free calcium with a peak a t 30 s followed by a sustained plateau phase. The initial peak is probably due to Ins(l,4,5)P3-mediated calcium mobilization from intracellular pools, since it coincides with the peak in Ins (1,4,5)P3 formation and is unaffected by removal of extracellular calcium with EGTA. By contrast the plateau phase was dependent on influx of calcium through calcium channels, in agreement with earlier findings (35). ATP responses were partially sensitive to treatment of cells with pertussis toxin, and these data indicate that ATP, via nucleotide receptors, activates phospholipase C via two types of G-proteins, one being sensitive to pertussis toxin treatment, the other not. A partial sensitivity of ATP receptor-mediated responses to treatment with PTX has been seen in other systems (40,41,43,44,46,47), but there are also systems where ATP-responses are insensitive to PTX (48). There is reason to assume that the PTX-insensitive G-protein belongs to the G,-family (49).
We found that adenosine A, receptors also couple to phospholipase C and the mobilization of intracellular calcium in DDT, MF-2 cells. A selective adenosine A1 receptor agonist, CPA, increased intracellular free calcium and the formation of Ins (1,4,5)P3 (EC5,, 4 and 10 nM, respectively). The effect was blocked by low concentrations of the selective adenosine A1 antagonist DPCPX, showing that the effect is mediated via typical adenosine A, receptors. This is in contrast to other reports of adenosine receptor-mediated activation of phospholipase C where absolute and relative potencies of agonists and antagonists have tended to be atypical (27-31) and even suggested novel types of adenosine receptors (30, 31). The adenosine-and CPA-induced Ins (1,4,5)P3 formation was smaller and peaked later than that induced by ATP. The CPA-induced rise in intracellular calcium had the same principal characteristics as the ATP response with an initial, Ins(l,4,5)P3-mediated peak followed by a sustained plateau phase that was abolished in the presence of EGTA. In agreement with lower Ins (1,4,5)P3 values, calcium increases were also lower when cells were stimulated with CPA than with ATP.
It seems likely that adenosine A, receptors are coupled to phospholipase C and to adenylylcyclase via the same Gprotein(s), since all the responses via phospholipase C were completely sensitive to treatment of cells with pertussis toxin. We have previously shown this to be the case for the coupling to adenylylcyclase (33). Using ADP-ribosylation and peptide antibodies we find evidence for two pertussis toxin substrates in DDT, MF-2 cells, Giz and Gi3 (39). There is evidence that either of these G-proteins may be involved in receptor-mediated activation of phospholipase C in HL60 cells (50), just as they may inactivate adenylylcyclase. A negative coupling to adenylylcyclase in combination with a positive coupling to phospholipase C has been shown for thrombin-(51) and angiotensin I1 receptors (52-54). We cannot, however, exclude the possibility that phospholipase C and adenylylcyclase are coupled to A, receptors preferentially via one or the other of the two PTX-sensitive G-proteins in DDT, .
Several studies have failed to demonstrate a direct activation of phospholipase C by adenosine receptors (16-24, 35, 55). Since these negative reports include an earlier study using the DDT, MF-2 cell line (35) and since the response we detected is a small one, it can not be excluded that lack of sensitivity of the methods used is one explanation for some of the earlier negative findings. The EC5,, value for Ins (1,4,5)P3 formation by CPA (10 nM) is higher than the E G O value (1.9 nM) for inhibition of CAMP formation and the KO at the high affinity binding site (0.45 nM) (33). It is possible that in order to observe an Ins (1,4,5)P3 response, a large number of adenosine receptors have to be activated, perhaps because the primary target of the A1 receptor-Giprotein cascade is not generally phospholipase C. This might also explain why in several cell systems the potency of adenosine analogues is atypically low.
A major finding of the present study is that the simultaneous activation of ATP receptors and A1 receptors by ATP and CPA caused a synergistic increase in the formation of Ins (1,4,5)P3 and mobilization of intracellular calcium. The effect was more than additive and CPA caused a concentration-dependent enhancement of ATP-induced Ins (1,4,5)P3 accumulation with an EC50 value (3 nM) typical for an adenosine A, receptor response. Thus, the present results are the most clearcut demonstration that adenosine, acting a t AI receptors, can increase intracellular Ins(1,4,5)P3 levels and elevate calcium per se and enhance the response mediated via other receptors. The synergy was lost after treatment of cells with pertussis toxin, indicating that the PTX-insensitive Gprotein is not a target for the adenosine AI receptor.
The synergy on Ins(1,4,5)P3 formation had functional importance for the mobilization of intracellular calcium, in agreement with a previously reported synergy between adenosine receptors and ATP receptors on the mobilization of intracellular calcium (55). However, in the previous study, no effect of adenosine receptor stimulation on phospholipase C was found, and the effect of a simultaneous activation of adenosine receptors and ATP receptors on the formation of inositol phosphates was not investigated. As mentioned in the introduction there are also several reports that adenosine analogues augment stimulation of phospholipase C by agonists to al-adrenergic, histamine HI, muscarinic, and GTP receptors. In these studies it has not been clearly shown that the effect was mediated via adenosine A, receptors. Furthermore, the synergistic effects were generally observed in long term incubations, and none of the authors have directly measured the calcium-mobilizing Ins( 1,4,5)P3 isomer.
ATP-induced Ins ( 1,4,5)P3 formation was attenuated in the presence of the adenosine A1 receptor antagonist DPCPX. Treatment of cells with adenosine deaminase mimicked this effect and abolished the DPCPX-mediated attenuation.
We therefore conclude that adenosine, possibly formed from ATP, can enhance the effect of ATP on Ins(1,4,5)P3 formation. Rapid breakdown of ATP by ecto-enzymes (10) has been shown to generate enough adenosine to interact with adenosine receptors and affect signal transduction (56). We show that ATP is metabolized to yield ADP, AMP, and adenosine. After 1 h, adenosine reached a concentration close to 200 nM in the incubation medium. However, in the immediate vicinity of the cell membrane the concentration of adenosine may be sufficient to activate adenosine receptors within 0.5-3 min, i.e. when the enhancement was observed. A local, rapid accumulation of adenosine, formed from ATP, has been postulated by other authors (56). We found no evidence for adenosine release from the cells when activated with ATP.
The fact that adenosine could be formed from ATP and interact with ATP raises a possibility that the observed receptor-receptor interaction may be physiologically important. It is known that ATP may act like a neurotransmitter and it might also affect other signaling functions (1,57). The signal molecule is rapidly degraded, leading to a very transient response. If a major metabolite acts synergistically one would expect a larger and more prolonged signal.
The mechanism behind the interaction is not known. The data presented here indicate that it is not dependent on CAMP, on membrane potential, on activation of phospholipase A' to generate arachidonic acid related factors, or on protein kinase C. It is not easy to understand how a second messenger generated by phospholipase C could mediate this response, since both receptors would generate the same messengers and a synergy was observed at maximal effective concentrations of either agonist. It is possible that the synergy is related to the fact that one of the receptors (the adenosine A1 receptor) is completely dependent on activation of a pertussis toxin-sensitive G-protein whereas the other receptor (the nucleotide receptor) is at least partly dependent on a pertussis toxin-insensitive pathway. This is supported by the finding that adenosine is also able to act in synergy with bradykinin, a response that is completely unaffected by pertussis toxin.' In view of the recent finding that G-protein /3,y subunits are able to enhance some effects mediated by asubunits on adenylylcyclase (58), it is tempting to speculate that the same may be true for some types of phospholipase C.
In summary, the present study shows that adenosine A1 receptors and nucleotide receptors (activated with ATP) independently mobilize intracellular calcium via the formation of Ins ( 1,4,5)P3 and that the two receptors act in synergy. It is shown that adenosine is formed from ATP, presumably by degradation via ecto-enzymes, and that this endogenous adenosine may act synergistically with ATP on Ins(1,4,5)P3 formation. We also show that in DDTl MF-2 cells, phospholipase C is coupled both to nucleotide receptors (via pathways involving one pertussis toxin-sensitive and one pertussis toxininsensitive G-protein) and to pertussis toxin-sensitive adenosine A1 receptors.