Introduction

Summary Introduction Procedures Results Discussion References

Despite its recent identification, the involvement of the A₃AR in several physiological effects of adenosine has been proposed; these include cardioprotection and neuroprotection from prolonged ischemia, bronchoconstriction, mast cell and eosinophil activation, and induction of hypotension (1-3). These phenomena are initiated by agonist binding to A₃AR proteins whose genes have been isolated from rat, sheep, rabbit, and humans (4-7). Despite the classification of these proteins as A₃ARs, the rat protein exhibits only a 70% identity with the other species homologues. This is reflected in notable pharmacological differences, in particular, a 100-1000-fold-lower affinity of the rat A₃AR for certain xanthine compounds compared with the recombinant human and sheep receptors (1, 5-7). A recent report demonstrated that the rat A₃AR mRNA is subject to an alternative splicing event within the coding sequence, resulting in the insertion of a 17-amino acid segment within the second intracellular loop (8). This report also stated that the human A₃AR message did not seem to undergo similar processing (8).

We demonstrated previously that prolonged agonist exposure of CHO cells expressing a recombinant rat A₃AR results in a desensitization of receptor function that is associated with the down-regulation of specific G protein subunits (9). Given the structural and pharmacological differences displayed by the rat and human A₃ARs, it is important to determine whether the desensitization mechanisms induced by agonist occupation of the rat A₃AR are unique to this species homologue or are characteristic of the other A₃ARs. In this study, we describe the effects of prolonged agonist exposure on CHO cells expressing a recombinant human A₃AR and demonstrate that prolonged agonist exposure not only results in receptor desensitization but also induces a sensitization of the stimulatory pathway of AC, which may have physiological and potential therapeutic implications.

	Experimental Procedures

Summary Introduction Procedures Results Discussion References

Materials. Radiochemicals were obtained from DuPont-New England Nuclear (Boston, MA). AB-MECA and IB-MECA were generously donated by Dr. Kenneth A. Jacobson (National Institutes of Health, Bethesda, MD). ¹²⁵I-AB-MECA was synthesized and purified as described previously (10). PTX and RO201724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone] were from Life Technologies (Grand Island, NY). Antibodies specific for CREB and Ser133-phosphorylated CREB were from New England Biolabs (Beverly, MA). Sources of other materials have been described previously (9-11).

Receptor cDNA constructs and expression. The human A₃ receptor cDNA was constructed by splicing together two partial cDNAs obtained from Dr. Sean Munro (Cambridge University, Cambridge, UK) to form a functional ORF. The open reading frame was subcloned into the BamHI site of the internal ribosome entry site element containing expression vector pCIN1 (12). The stable transfected cell line 93.1.1 was generated in the following manner: 50 µg DNA (pCIN1/hA3) was linearized in 50 µl with SspI and sterilized by adding 50 µl of phenol/chloroform/isoamyl-alcohol (25:24:1) to the 50-µl DNA sample in a phase-lock tube. Contents were mixed, and the tube was spun for 30 sec. Chloroform/isoamyl-alcohol [50 µl (24:1)] was added to the upper aqueous phase, and the tube was respun. The upper aqueous phase containing the 50 µg of DNA was removed asceptically. The CSH host line 53.1.12 was maintained in Dulbecco's modified Eagle's medium/Ham's F-12 medium (1:1) with 15 mM HEPES (no. 42400; Life Technologies), 10% (v/v) fetal calf serum, and 500 µg/ml Hygromycin and passaged when 50% confluent. The cells were resuspended at 1 × 10⁷/ml in the Dulbecco's modified Eagle's medium/Ham's F-12 medium. Then, 450 µl of cells plus 50 µl of sterile linearized DNA were electroporated with a single pulse in a Gene Pulsar I apparatus (Biorad, Melville, NY) using a 0.4-cm cuvette at 960 µF and 280 V. The time constant was 20 msec. The cells were diluted immediately into 30 ml of media and cultured at 37°. Media was changed every 3 days until day 12, on which the cells were dilution cloned. A single clone was chosen empirically from the 12 tested on the basis of (a) a 4-fold response to 10⁵ M forskolin (water-soluble derivative, no. 344273; Calbiochem, San Diego, CA) in terms of secreted placental alkaline phosphatase production and (b) a dose-responsive reduction in the latter by NECA. Cells were maintained in Ham's F-12 media supplemented with 10% (w/v) fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin as described previously (9-11).

Antibodies and immunoblotting. Antibodies versus specific G protein subunits were generously donated by Dr. Tom Gettys (Medical University of South Carolina, Charleston, SC). The generation and specificities of these antisera have been described previously (13, 14). The primary antibodies used in this study were 982 (1:8,000 dilution of serum) for detection of G_i-2, 977 (1:8,000 dilution of serum) for detection of G_i-3, 951 (1:16,000 dilution of serum) for detection of G_s, 457 (1:1,000 dilution of protein A affinity-purified IgG) for detection of G_q and G₁₁, and 987 (1:8,000 dilution of serum) for detection of  subunits. Membranes were prepared from confluent T-75 flasks as we described previously and aliquoted for storage at 80° (9). After quantification of protein content according to the method of Bradford (15), equivalent amounts of membrane protein (typically 15 µg) were resolved by discontinuous SDS-PAGE on 10% (w/v) polyacrylamide resolving gels using a BioRad (Hercules, CA) Mini Protean 2 system. After transfer to a nitrocellulose membrane, nonspecific protein binding sites were blocked by a 60-min room temperature incubation in blocking buffer [5% (w/v) skim milk powder in phosphate-buffered saline containing 0.2% (v/v) Triton X-100 and 0.02% (w/v) thimerosal]. Nitrocellulose membranes were then incubated with the appropriate dilution of primary antibody in fresh blocking buffer overnight at 4°. After removal of antiserum and extensive washing with three changes of blocking buffer, membranes were incubated with a 1:5,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG in a high-detergent skim milk solution. The series of washes described above was then repeated and followed by two additional washes in phosphate-buffered saline before visualization of reactive proteins by an enhanced chemiluminescence protocol. Quantification of immunoblots was by densitometric scanning as we described previously (9).

Radioligand binding and AC assays. Radioligand binding and AC assays were performed and analyzed exactly as we described previously, except that for AC assays, 20 µM RO201724 was used as the phosphodiesterase inhibitor instead of papaverine (10, 11).

[³H]Leucine incorporation. Triplicate wells of transfected CHO cells in six-well dishes were incubated for 6 hr in regular media supplemented with 1 µCi/well of [³H]leucine with or without 30 µg/ml cycloheximide. Incubations were terminated by placing the cells on ice, washing with ice-cold phosphate-buffered saline, and solubilizing the cell monolayers in detergent buffer as described above. An equal volume of 72% trichloroacetic acid was added to solubilized extracts, and the resulting precipitates were collected by microcentrifugation. Pellets were solubilized in 1 M sodium hydroxide, and [³H]leucine incorporation was determined by liquid scintillation counting.

	Results

Summary Introduction Procedures Results Discussion References

Functional desensitization of the human A₃AR. The cell line used for these experiments bound the A₃AR agonist radioligand ¹²⁵I-AB-MECA with high affinity (K_d = 1.24 ± 0.41 nM; four experiments) and exhibited B_max values of 0.38-0.68 pmol/mg of protein (four experiments). Radioligand binding experiments demonstrated that exposure of transfected cells to 10 µM concentration of the AR agonist NECA for 20 hr resulted in a 73 ± 7% reduction in B_max versus vehicle-treated controls (p < 0.05; four experiments) without significantly affecting the K_d values (1.24 ± 0.41 nM for control and 1.42 ± 0.13 nM for agonist-treated cells; four experiments) (Fig. 1A). This was associated with a functional desensitization of A₃AR signaling as determined by analysis of IB-MECA-mediated inhibition of forskolin-stimulated AC activity in isolated membranes, with maximal inhibition falling from 57 ± 8% to 14 ± 10% after a 20-hr agonist treatment (p < 0.05 versus vehicle-treated controls; three experiments) and the IC₅₀ value for IB-MECA increasing from 19 ± 4 to 95 ± 60 nM (p < 0.05 versus vehicle-treated controls; three experiments) (Fig. 1B). Therefore, under conditions in which agonist treatment results in a profound reduction in agonist radioligand binding at the A₃AR, the signaling capacity of the A₃AR undergoes functional desensitization.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effects of chronic exposure to NECA on A3AR function. A, Transfected CHO cells were incubated in the absence (CONTROL) or presence (TREATED) of 10 µM NECA for 20 hr at 37°. After extensive washing of cell monolayers, membranes were prepared for saturation radioligand binding experiments with increasing concentrations of 125I-AB-MECA and analysis as described in Experimental Procedures. Nonspecific binding was assessed by the inclusion of 50 µM NECA. Inset, Scatchard transformations of the specific binding data from this experiment. This is one of four experiments that produced similar results. B, Membranes from transfected CHO cells treated as described in A were assayed for AC activity in the presence of 5 µM forskolin and increasing concentrations of IB-MECA as described in the Experimental Procedures. This is one of three experiments that produced similar results.

Effects of agonist treatment on G protein levels. We demonstrated previously that prolonged agonist exposure of the rat A₃AR expressed in CHO cells results in the down-regulation of specific G protein subunits (9). To determine whether chronic exposure of the human A₃AR to agonist could mediate similar effects, membranes from transfected cells were analyzed by comparative immunoblotting after treatment with or without 10 µM NECA for 20 hr (i.e., conditions that produced a desensitization of human A₃AR function) (Fig. 1). Immunoblotting with antisera specific for G_i-2, G_i-3, and  subunits common to multiple G proteins demonstrated that each of these proteins were down-regulated by agonist treatment (Fig. 2, A-C, and Table 1). Moreover, the altered expression levels of each of these proteins did not reflect a nonspecific global change in the total pool of cellular G protein subunits in that levels of G_s and G_q+11 subunits were unaffected (Fig. 2, D and E, and Table 1).

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Effects of chronic exposure to NECA on G protein expression. Aliquots (15 µg) of membranes prepared from transfected CHO cells incubated in the absence (C) or presence (T) of 10 µM NECA for 20 hr at 37° were subjected to SDS-PAGE and immunoblotting with antisera specific for Gi-2 (A), Gi-3 (B),  subunits (C), Gs (D), and Gq+11 subunits (E) as described in Experimental Procedures. Data pooled from three separate comparisons for each G protein subunit are given in Table 1.

View this table: [in this window] [in a new window]   TABLE 1 Agonist regulation of G protein expression in CHO cells expressing the human A3AR CHO cells expressing the human A3AR were treated with or without 10 µM NECA for 20 hr at 37° before membrane preparation and comparative immunoblotting as described in Experimental Procedures. Under these conditions, Gq and G11 comigrate, and therefore the signal observed on immunoblots represents a composite signal for these proteins. Data are presented as mean ± standard error for three experiments with the signal in untreated controls set at 100%.

Effects of A₃AR agonist treatment on AC stimulation. Under conditions that produced A₃AR desensitization, a ~2-fold increase was observed in the stimulation of AC activity obtained in the presence of GTP with or without forskolin (Table 2). This effect required the presence of a functional A₃AR because a 20-hr exposure of nontransfected CHO cells to 10 µM NECA resulted in a 6 ± 17% change [p > 0.05 (NS); three experiments] in GTP-stimulated AC activity in subsequently isolated membranes. The changes in AC stimulation were not reflected in an increased activity of AC catalytic units in these membranes because when assays were performed in the presence of forskolin and manganese chloride to uncouple the catalytic units from G protein regulation (16), no difference in activity was found between membranes from control cells and those from agonist-treated cells (Table 2). This initially suggested that the down-regulation of G_i proteins induced on agonist exposure removed a low-level tonic inhibitory effect of G_i on AC activity in these cells. Moreover, if this were the case, it would be expected that this effect could be mimicked by treatment of transfected cells with PTX, which would inactivate G_i by catalyzing its ADP-ribosylation as opposed to the down-regulation induced on chronic agonist exposure. Exposure of transfected cells with sufficient PTX to abolish IB-MECA-mediated inhibition of forskolin-stimulated AC activity failed to result in a significant increase in GTP-stimulated AC activity (Fig. 3). However, PTX treatment completely attenuated the ability of NECA to sensitize AC activity (Fig. 3). Therefore, the ability of chronic A₃AR activation to sensitize AC stimulation is not a result of G_i down-regulation and requires a functional A₃AR/G_i signaling system to be manifested.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Effect of PTX treatment on agonist-induced AC sensitization. Transfected CHO cells were incubated in the presence or absence of 10 µM NECA and 100 ng/ml PTX for 20 hr at 37° as indicated. Membranes were then prepared for assay of AC activity in the presence of 5 µM GTP as described in Experimental Procedures. Data are pooled from three experiments. The 5 µM GTP-stimulated activity in membranes from cells treated without either NECA or PTX was 2.00 ± 0.26 pmol/min/mg. The percent inhibition of 5 µM forskolin-stimulated AC activity elicited by 1 µM IB-MECA for each treatment condition was 34 ± 4% (Vehicle), 9 ± 3% (NECA), 0 ± 1% (PTx + vehicle), and 1 ± 1% (PTX + NECA). *, Significant difference (p < 0.05) from the specific activity achieved in membranes from cells treated without agonist or PTX.

s

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Time course of agonist-induced AC sensitization. A, Transfected CHO cells were treated with 10 µM NECA at 37° for the indicated times before membranes preparation and assay of AC activity in the presence of 5 µM GTP as described in Experimental Procedures. Data are pooled from three experiments. Inset, immunoblot for Gs subunits in 15-µg aliquots of membranes prepared from transfected CHO cells incubated at 37° with 10 µM NECA for the indicated times. B, Transfected CHO cells were incubated in the presence or absence of 10 µM NECA and 30 µg/ml cycloheximide (CHX) for 6 hr at 37° as indicated. Membranes were then prepared for assay of AC activity in the presence of 5 µM GTP as described in Experimental Procedures. Data are pooled from three experiments. The 5 µM GTP-stimulated activity in membranes from cells treated without either NECA or CHX was 2.28 ± 0.26 pmol/min/mg. The percent inhibition of 5 µM forskolin-stimulated AC activity elicited by 1 µM IB-MECA for each treatment condition was 54 ± 3% (Vehicle), 29 ± 7% (NECA), 56 ± 4% (CHX + Vehicle), and 30 ± 3% (CHX + NECA). *, Significant difference (p < 0.05) from the specific activity achieved in membranes from cells treated without agonist or CHX.

Effects of agonist treatment on stimulation of CREB phosphorylation. Given that the increase in GTP-stimulated AC activity induced by chronic A₃AR activation was only 1.5-2-fold over control, it was important to determine whether this effect had consequences for long term adaptation in intact cells. This was determined by assessing the effect of agonist pretreatment on the subsequent ability of a submaximal concentration of forskolin to stimulate phosphorylation of CREB, which is regulated by cAMP in part through a well-characterized phosphorylation of Ser133 that can be detected immunologically (17). In vehicle-treated controls, 1 µM forskolin stimulated CREB phosphorylation by 3.5 ± 1.7-fold over basal after a 30-min incubation (Fig. 5A). In cells pretreated with 10 µM NECA for 20 hr, 1 µM forskolin produced levels of CREB phosphorylation ~220 ± 40% greater than that seen in vehicle-treated cells (p < 0.05; three experiments) without changing the basal level of phosphorylation (Fig. 5A). A similar pattern of elevated forskolin-stimulated phosphorylation was observed for ATF-1, which cross-reacts with this antibody preparation (Fig. 5A). The changes in CREB phosphorylation were not the result of a parallel change in the levels of CREB expression, as determined by immunoblotting the same extracts with an antibody that recognizes CREB regardless of its phosphorylation status (Fig. 5B). In contrast, similar pretreatment of nontransfected cells failed to produce augmentation in the subsequent ability of forskolin to increase CREB or ATF-1 phosphorylation (Fig. 5A).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Effect of chronic agonist exposure and subsequent withdrawal on CREB phosphorylation. A, Nontransfected and transfected CHO cells were incubated for 20 hr at 37° in the absence (Vehicle) or presence of 10 µM NECA before washing of cell monolayers with prewarmed phosphate-buffered saline and a subsequent 30-min incubation at 37° in the presence or absence of 1 µM forskolin (Fsk) as indicated. Detergent extracts were then prepared for immunoblotting with antibodies versus phosphorylated CREB as described in Experimental Procedures. Quantitative data from three experiments are presented in Results. B, Transfected CHO cells were incubated without (Vehicle) or with 10 µM NECA for 20 hr at 37° before agonist washout and a further 30-min stimulation with 1 µM forskolin (Fsk) as indicated. Detergent extracts were then prepared for immunoblotting with antibodies versus CREB as described in Experimental Procedures. This is one of three experiments that produced similar results.

	Discussion

Summary Introduction Procedures Results Discussion References

On the binding of an agonist ligand to a G protein-coupled receptor, multiple cellular mechanisms may be invoked to control both the signal emanating directly from the receptor and the responsiveness of receptors that regulate other signaling pathways (18). We demonstrated that exposure to agonist of cells expressing the human A₃AR affects both the inhibitory and stimulatory arms of AC regulation. Consistent with our previous report on the desensitization of the rat A₃AR (9), prolonged agonist exposure resulted in a profound functional desensitization that was associated with a reduction in the number of high affinity agonist binding sites detectable by radioligand binding. The loss of high affinity binding sites is indicative of a reduction in the number of signaling-competent receptor/G protein complexes and may be due to any of several reasons. Because the human A₃AR is capable of inhibiting AC activity in a PTX-sensitive manner, which is indicative of an ability to couple productively with G_i proteins, the large reductions in the levels of membrane-associated G_i-2 and G_i-3 may be partly responsible for the loss in high affinity binding observed on agonist exposure. On the basis of observations made with other G protein-coupled receptors, it was also possible that the A₃AR protein was down-regulated in response to agonist. In the absence of either a high affinity antagonist radioligand or an antibody of sufficiently high sensitivity to assess A₃AR expression, we cannot examine this possibility.

A previously unappreciated consequence of chronic A₃AR activation was the time-dependent onset of AC sensitization. Specifically, agonist treatment enhanced the stimulation of AC activity elicited by GTP in the absence or presence of forskolin but not in the presence of forskolin and manganese chloride. This phenomenon was not a reflection of a diminution in G_i function due to down-regulation of G_i subunits because abolition of G_i function with PTX failed to mimic the effect of chronic agonist exposure, thereby implicating the stimulatory pathway as the locus for the altered regulation of AC. We also observed that the ability of prolonged agonist treatment to sensitize AC stimulation is not restricted to the human A₃AR; similar effects on AC regulation are involved through chronic agonist exposure of the rat A₃AR.²

Forskolin binds to and activates all nine of the AC isoforms cloned to date, although there is some evidence for isoform-specific variations in the levels of activation that can be achieved (19-21). However, the interaction is greatly enhanced in the presence of G_s, a property used by several investigators to quantify G_s/AC coupling in intact cells and isolated membranes (20, 22, 23). Therefore, the AC activity achieved in the presence of forskolin and GTP reflects the stimulation of G_s-coupled AC proteins. In contrast, the addition of manganese to the assay uncouples AC from G protein regulation; therefore, the activity observed with forskolin and MnCl₂ reflects the activity of AC catalytic units independent of G protein function (16). Hence, the lack of any enhancement of AC stimulation observed with forskolin in the presence of MnCl₂ suggests the functioning of the G_s/AC complex was specifically enhanced by agonist pretreatment. An increase in the levels of membrane-associated G_s subunits was not responsible because over the time course in which sensitization of AC was observed, no significant change in the expression of membrane-associated G_s subunits could be detected. Moreover, several studies addressing the consequences of overexpression of G_s on AC regulation found that even high degrees of overexpression of this protein have little effect on the maximal AC stimulation that can be achieved (24, 25). Therefore, agonist treatment must either increase the ability of G_s to stimulate AC and/or increase the ability of AC catalytic units to respond to activated G_s. Although the mechanism for this phenomenon remains obscure, it does not seem to involve de novo protein synthesis, because a maximally effective concentration of cycloheximide was without effect, or a slow-onset activation of specific AC isoforms by PKC-mediated phosphorylation, because maximally effective concentrations of a selective PKC inhibitor failed to block the sensitizing effect of agonist pretreatment.

The ability of chronic treatment with inhibitory agonists to sensitize the stimulatory pathway of AC has been described for several G protein-coupled receptors, including dopamine D₂ and muscarinic acetylcholine m2 receptor subtypes as well as the rat adipocyte A₁ adenosine receptor (26-28). However, most research has been focused on the ability of morphine, acting at the µ-opioid receptor, to sensitize AC activity both in cultured cells (29-32) and in specific regions of the brain because the sensitization phenomenon is thought to play a crucial role in the behavioral changes associated with opiate withdrawal in humans (33). The sensitivity to PTX, insensitivity to cycloheximide, and time course of onset of sensitization we described here are essentially the same properties as the morphine-induced sensitization of AC induced by a recombinant µ-opioid receptor expressed in CHO cells (30). Further experiments by the same group demonstrated that although AC I, V, VI, and VIII can be sensitized in response to prolonged µ-opioid receptor activation, AC II, III, IV, and VII are not sensitized under the same experimental conditions (31, 32). Because we have shown that the specific activity of G protein-uncoupled AC catalytic units is unaffected by conditions in which AC sensitization is observed in response to A₃AR activation, it may be that AC isoform specificity for this phenomenon reflects a sensitivity to an unknown regulatory factor capable of modulating G_s/AC interactions.

In contrast to the similarities with the µ-opioid receptor system, our data appear distinct from that of Thomas and Hoffman (27) because half-maximal sensitization by agonist occupation of the muscarinic m2 receptor in their transiently transfected human embryonic kidney 293 cell system occurred ~5 min after agonist exposure rather than the 2-3 hr we and Vogel et al. (30) observed in CHO cells (30). Therefore, it seems likely that the phenomenon we observed in CHO cells represents one of several potential AC sensitization mechanisms that may exist, whose contribution to the overall effect may vary in a cell type-specific manner. However, an involvement of G protein subunits in mediating AC sensitization in both of these systems has been proposed, although in each case this effect on AC seems to be indirect (i.e., not the result of a direct interaction between subunits and the classic -stimulated AC isoforms, AC II and IV) because ACV, which is not activated directly by subunits, can still be sensitized in a manner blocked by overexpression of scavenger proteins (27, 31). The relevance of these findings to the results presented here remains to be determined.

Because the A₃AR-induced sensitization of GTP-stimulated AC activity in isolated membranes was relatively modest (1.5-2.5-fold), it was important to determine whether this phenomenon had any effect on cAMP-regulated events in an intact cell. The phosphorylation of CREB was chosen as a measure of significance at the intact cell level because long term adaptive changes in response to extracellular stimuli typically result in altered patterns of gene expression, which requires mobilization of appropriate transcription factors. Moreover, CREB is expressed constitutively and is predominantly regulated by a well-characterized cAMP-dependent protein kinase-catalyzed phosphorylation event that is readily detectable (17). The observations that chronic A₃AR activation resulted in increased forskolin-stimulated phosphorylation of CREB (and ATF-1) on agonist removal and that this phenomenon is not observed in nontransfected cells suggest strongly that the A₃AR-induced sensitization of AC detectable in isolated membranes reflects the induction of an important adaptive process that may have consequences for cellular regulation of gene transcription.

Finally, the sensitization of AC reported here may provide a molecular basis for the observation that for several adenosine receptor-mediated events, acute agonist exposures produce opposite effects to those of chronic agonist treatments. This "effect inversion" phenomenon has been observed with several AR-mediated physiological processes, including neuroprotection from ischemia, which may involve the A₃AR (3, 34). Specifically, acute preischemic activation of gerbil A₃ARs with the A₃AR agonist IB-MECA increases postischemic cerebral damage and mortality, whereas chronic treatment with IB-MECA reduces these parameters after an ischemic insult (2). The ability of chronic A₃AR activation to sensitize signal transduction pathways diametrically opposed to those induced after acute exposure could explain this phenomenon. Interestingly, a recent study has shown that enhanced survival of dentate granule cells after hypoxic-ischemic injury in rats is associated with elevated levels of phosphorylated CREB compared with CA1 pyramidal cells, which readily undergo programmed cell death after an ischemic insult (35). Any potential relationship between these observations made in intact animal models and the A₃AR-mediated sensitization of AC we report here remains to be elucidated.