1Department of Physiology, School of Pharmacy, Universidad del País Vasco, Bilbao; and 2Department of Biochemistry and Molecular Biology, School of Medicine, Universidad de Alcalá, Alcalá de Henares, Spain
Submitted 5 March 2004 ; accepted in final form 19 October 2004
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ABSTRACT |
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potassium currents; compartmentalization
Because Ito is the main current responsible for the duration of the action potential in the rat (2, 12) and for phase 1 in humans (46), the magnitude of this current is highly regulated. In rat ventricular myocytes, the Ito channel seems to be a heterotetramer composed of cloned voltage-dependent K+ channels Kv4.3 and Kv4.2 (39, 44). The amino acid sequences of both Kv4.2 and Kv4.3 reveal phosphorylation consensus sites for protein kinase A (PKA), protein kinase C (PKC), ERK, and CaMKII (13, 31, 38). In fact, Kv4.3 is regulated in vivo by PKC (34); native Ito and expressed Kv4.2 and Kv4.3 are phosphorylated by CaMKII (11); and Kv4.2 is phosphorylated by PKA and ERK both in vitro and in vivo (1, 3).
In the heart, the most extensively documented biochemical responses to 1-adrenergic stimulation is G
q activation of phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate, which gives rise to 1,2-diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3), as well as to PKC activation by these second messengers (9, 34).
In rat ventricular cells, suppression of a Ca2+-independent, voltage-activated, transient K+ current in response to the activation of 1-adrenoceptors has been reported (4). These authors postulated that the effect might be mediated via activation of PKC. Since the publication of that study, different authors have corroborated or rejected this pathway (8, 32, 36, 43). Our results exclude this classic
1-adrenergic pathway. In the present study, we found that in isolated rat cardiac myocytes,
1-adrenoceptor stimulation activates G
s and inhibits the native Ito via a cAMP/PKA-mediated signaling pathway.
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MATERIALS AND METHODS |
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Cell isolation. Sprague-Dawley rats (200220 g) were anesthetized with chloral hydrate (3 ml/kg body wt, delivered intraperitoneally) and killed by cervical dislocation. The hearts were removed and perfused with a Tyrode solution containing (in mmol/l) 118 NaCl, 5.4 KCl, 24 NaHCO3, 1.02 MgCl2, 1.8 CaCl2, 0.42 NaH2PO4, 12 dextrose, and 20 taurine, bubbled with 95% O2-5% CO2, pH 7.4, at 37°C, followed by the same solution without Ca2+ and then by the same nominally Ca2+-free solution containing collagenase type I (0.5 mg/ml) and protease type XIV (0.03 mg/ml). The hearts were finally perfused with a KB solution (25) containing (in mmol/l) 10 taurine, 70 glutamic acid, 0.5 creatine, 5 succinic acid, 10 dextrose, 10 KH2PO4, 20 KCl, 10 HEPES-K+, and 0.2 EGTA-K+, adjusted to pH 7.4 with KOH. Single cells were obtained using mechanical agitation.
Current recordings.
Ito is rapidly activated, so experiments were performed at room temperature (2022°C) to isolate peak current from capacitive current. Ionic currents were recorded using the whole cell variation of the patch-clamp technique (21) with an Axopatch 200B patch-clamp amplifier (Axon Instruments, Union City, CA). Recording pipettes were obtained from borosilicate tubes (Sutter Instruments, Novato, CA) and had a tip resistance of 13 M when filled with the internal solution containing (in mmol/l) 80 L-aspartic acid (K+ salt), 10 KH2PO4, 1 MgSO4, 50 KCl, 5 HEPES-K+, 3 ATP-Na2, and 10 EGTA-K+, adjusted to pH 7.3 with KOH.
The bathing solution contained (in mmol/l) 86 NaCl, 1 MgCl2, 10 HEPES-Na+, 4 KCl, 0.5 CaCl2, 2 CoCl2, 12 dextrose, and 50 tetraethylammonium (TEA)-Cl, adjusted to pH 7.4 with NaOH. Ito was recorded applying depolarizing pulses to +50 mV, starting from a holding potential of 60 mV. The TEA-resistant, time-independent Iss was digitally subtracted. Peak Ito were normalized to cell capacitance and expressed in picoamperes per picofarad.
Chemicals.
Norepinephrine (NE), phenylephrine (PE), isoproterenol, methoxamine, neomycin, chloroethylclonidine, collagenase, and protease were obtained from Sigma Chemical (St. Louis, MO). C6-ceramide (D-erythrosphingosine, N-hexanoyl), calmidazolium, H89, forskolin, 2',5'-dideoxyadenosine (DDA), bisindolylmaleimide I, 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), haloenol lactone suicide substrate (HELSS), 8,8'-[carbonyl-bis(imino-3,1-phenylene)]-bis-(1,3,5-naphthalenetrisulfonic acid) (NF023), 4,4',4'',4'''-{carbonyl-bis[imino-5,1,3-benzenetriyl-bis(carbonylimino)]}tetrakis-(benzene-1,3-disulfonate) (NF449), and G protein antagonist 2A were obtained from Calbiochem (San Diego, CA). Prazosin, propranolol, 5-methylurapidil, and BMY-7378 were purchased from Tocris (Ellisville, MO). Because not all of the enzyme inhibitors used are fully specific, we blocked every intracellular pathway at least at two different steps, usually at the protein kinase step and at the protein kinase activator step (i.e., PKC with bisindolylmaleimide I, PLC with neomycin and Gq with G protein antagonist 2A).
Membrane isolation. All procedures were performed at 4°C in a homogenization buffer (HF) containing 20 mmol/l Tris·HCl, pH 7.4, 1 mmol/l EDTA, and 2.5 µl/ml of the Sigma protease inhibitor cocktail (Sigma Chemical). Myocytes were homogenized for 1 min on ice. Nuclei and debris were pelleted by performing centrifugation at 500 g for 10 min. The supernatant was centrifuged at 40,000 g for 30 min. The pellet was resuspended in HF and centrifuged again at 40,000 g for 30 min. The final pellet was stored at 80°C. The protein content of all preparations was determined using the Bradford method (7).
Western blotting. Myocytic membranes were fractionated on 10% SDS-polyacrylamide gels and transferred to PVDF membranes (Immobilon P; Millipore, Billerica, MA). PVDF membranes were blocked in a solution (50 mmol/l Tris·HCl, pH 7.5, 150 mmol/l NaCl, and 0.05% Tween 20) containing 3% BSA. Blots were incubated with a primary antibody anti-phospho-Ser/Thr (1:1,000 dilution; Cell Signaling Technology, Beverly, MA) and donkey anti-rabbit IgG (1:5,000 dilution; Amersham Biosciences, Piscataway, NJ). Blots were developed using enhanced chemiluminescence (ECL; Amersham Biosciences). Coomassie blue staining and anti-Kv4.3 antibody (1:200 dilution; Chemicon International, Temecula, CA) were used as controls for equal protein loading.
Adenylyl cyclase assay. Myocyte membranes were incubated with 1.5 mmol/l ATP, 5 mmol/l MgSO4, 10 µmol/l GTP, an ATP regenerating system (7.5 mg/ml creatine phosphate and 1 mg/ml creatine kinase), 1 mmol/l 3-isobutyl-1-methylxanthine (IBMX), 0.1 mmol/l PMSF, 1 mg/ml bacitracin, 1 mmol/l EDTA, and test substances (30 µmol/l PE and 100 µmol/l forskolin) in 100 µl of 25 mmol/l triethanolamine-HCl buffer, pH 7.4. After a 30-min incubation at 30°C, the reaction was stopped by boiling the mixture. Then, 200 µl of an alumina slurry (0.75 g/ml in triethanolamine-HCl buffer) were added, and the suspension was centrifuged. The supernatant was obtained to assay the cAMP levels using the method of Gilman (19).
In another group of experiments, freshly isolated myocytes were incubated with PE 30 µmol/l for 15 min at 37°C. The reaction was stopped by adding a cold buffer containing 6 mmol/l EDTA, 30 mmol/l MgSO4, 6 mmol/l IBMX, 0.6 mmol/l PMSF, and 6 mg/ml bacitracin in 25 mmol/l triethanolamine-HCl buffer, pH 7.4. The cells were pelleted and resuspended in the same buffer without PE. The cell suspension was homogenized for 1 min on ice and centrifuged at 48,000 g for 15 min. Subsequently, 200 µl of the alumina solution were added to the supernatant, and cAMP was assayed using the same method. When used, 1-adrenoceptor subtype-selective blockers were added to the cell suspension 15 min before PE.
Coimmunoprecipitation.
Myocyte membranes were solubilized in radioimmunoprecipitation assay (RIPA) buffer (50 mmol/l Tris·HCl, pH 7.4, 150 mmol/l NaCl, 1 mmol/l EDTA, 1% Igepal, 1% sodium deoxycholate, and 2.5 µl/ml of the Sigma protease inhibitor cocktail). Membrane proteins (150 µg) were treated with 30 µmol/l PE, 10 µmol/l guanosine 5'-O-(3-thiotriphosphate) (GTPS), or 10 µmol/l NF449 for 1 min at 37°C. The reaction was stopped by addition of cold buffer, and membranes were centrifuged at 48,000 g for 15 min. The pellet was resuspended in 150 µl of RIPA buffer and incubated for 1 h at 4°C. Samples were cleared by centrifugation at 15,000 g for 25 min and supernatants were incubated with 2 µg of the anti-G
s antibody (Santa Cruz Technologies, Santa Cruz, CA). Protein G-agarose at 50% concentration (100 µl) was added, and the mixture was incubated for 3 h at 4°C. The beads were pelleted and washed three times in RIPA buffer. The bound proteins were eluted using 50 µl of SDS sample buffer and separated on 9% SDS-polyacrylamide gels. Western blot was performed with an anti-
1-adrenoceptor antibody (1:400 dilution; Oncogene, San Diego, CA) and goat anti-rabbit secondary antibody (1:4,000 dilution; Santa Cruz Technologies).
Data analysis. Current traces were analyzed using the Clampfit program of pClamp software (Axon Instruments). Enzyme inhibitors or activators were added to the pipette solution. After the patch rupture, currents were allowed to stabilize and control records were made. After this stabilization period, myocytes were exposed to NE or PE. Student's paired t-test was used for statistical analysis. Western blot bands were analyzed with Scion Image software (Scion, Frederick, MD). Student's t-test (for unpaired data) or Dunnett's test was used for statistical analysis when two or more groups were compared. The data are expressed as means ± SE. P < 0.05 was considered statistically significant.
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RESULTS |
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PLC and PKC modulation does not affect PE-induced Ito inhibition.
In many cell types, 1-adrenergic stimulation activates PLC, which leads to PKC activation. To explore this classic pathway, we blocked both PLC and PKC with neomycin (150 µmol/l) and bisindolylmaleimide I (1 µmol/l) (Fig. 3). Exposure of myocytes to PE in the presence of either neomycin or bisindolylmaleimide I also inhibited the Ito to an extent similar to that observed with PE alone. These results exclude PLC and most PKCs as members of the pathway involved in Ito reduction.
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PE-induced Ito inhibition is not mediated by Ca2+/calmodulin kinase II.
To explore the possibility that CaMKII might mediate the effects of the 1-agonists, we used the calmodulin antagonist calmidazolium (10 µmol/l). When administered in the presence of calmidazolium, PE caused current inhibition compared with control that was similar to that induced by PE in the absence of antagonist (Fig. 3).
Possible role of atypical PKC in 1-mediated Ito inhibition.
Although bisindolylmaleimide I (1 µmol/l) inhibits the classic and novel PKC subfamilies, the atypical PKC subfamily requires a drug concentration two orders of magnitude higher (30). The atypical PKC-
isoform, which is responsive to arachidonic acid, may exist in cardiac tissue. The
-subunits of G proteins can activate PLA2 (26) and give rise to the PKC-
activator arachidonic acid (28). In subsequent experiments, we used the PLA2 blocker HELSS (10 µmol/l) and a higher bisindolylmaleimide I concentration (100 µmol/l) to inhibit PKC-
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As shown in Fig. 3, PE in the presence of HELSS inhibited Ito to the same extent as did PE alone. To the contrary, administration of PE plus bisindolylmaleimide I (100 µmol/l) led to a slight but nonsignificant inhibition of Ito, suggesting that current reduction could be mediated by PKC-.
However, this high bisindolylmaleimide I concentration also inhibited the cAMP-dependent PKA. Therefore, to ascertain which protein kinase was mediating Ito inhibition, the following experiments were performed.
Involvement of the cAMP/PKA system in the Ito response to PE.
To determine whether PKA was involved in Ito inhibition, we used the specific PKA inhibitor H89 (5 µmol/l) as well as the adenylyl cyclase inhibitor DDA (300 µmol/l). PE did not inhibit Ito in the presence of either H89 or DDA (Fig. 4), suggesting that the effect of the 1-agonists on Ito inhibition was mediated by the cAMP-dependent PKA. In addition, we used the adenylyl cyclase activator forskolin (100 µmol/l) and the cAMP analog 8-BrcAMP (10 µmol/l) to directly activate PKA. Either forskolin alone or forskolin plus PE caused a similar and statistically significant inhibition of Ito. Likewise, 8-BrcAMP inhibited Ito to a similar extent when administered alone or together with PE. These results show that the effects of forskolin, 8-BrcAMP, and PE on Ito are similar and nonadditive, indicating that these components are involved in a common pathway (Fig. 4).
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The Ito reduction is an 1-specific effect.
Although PE is an
1-agonist, at very high concentrations, it can also activate
-adrenoceptors, which are classically coupled to cAMP/PKA activation. However, PE in combination with propranolol (1 µmol/l) reduced the Ito amplitude to the same extent as that observed in the absence of the
-antagonist. Similarly, we found that methoxamine (30 µmol/l), an
1-agonist that is more specific than PE, reduced Ito in the presence of propranolol. Finally, we tested the effect of the specific
-adrenoceptor agonist isoproterenol (1 µmol/l), which did not affect the Ito amplitude (Fig. 5). These results, together with those shown in Fig. 1, reveal that PE reduces Ito amplitude by interacting exclusively with
1-adrenoceptors.
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The next step was to identify the 1-adrenoceptor subtype involved in cAMP production by using the
1A-blocker 5-methylurapidil (15 nmol/l), the
1B-blocker chloroethylclonidine (10 µmol/l), and the
1D-blocker BMY-7378 (1 nmol/l). The cells were incubated with the different subtype-selective blockers, and 15 min later, PE (30 µmol/l) was added and cAMP production was measured. As shown in Fig. 6B, both
1A- and
1B-adrenoceptor subtypes were coupled to cAMP production, whereas the
1D-adrenoceptor was not.
Most of the known compartmentalization mechanisms involve the cytoskeleton, so we tested the effects of colchicine, a microtubule-disrupting agent. As shown in Fig. 6, C and D, when myocytes were pretreated with colchicine (5 µmol/l), PE had no effect on Ito amplitude, supporting the hypothesis regarding the compartmentalization of the signaling pathway.
1-Adrenoceptor activates G
s.
1-Adrenoceptors are members of the G protein-coupled receptor family, which is classically coupled with G
q dissociation. Although, as described above, neither PLC nor PKC is involved in PE-induced Ito reduction, we examined the role of G
q using G protein antagonist 2A (a peptide that selectively blocks the receptor-mediated G
q activation). PE reduces Ito by enhancing cAMP levels, which activate PKA. However, several G proteins could be involved in this process. Thus the G
s subtype directly enhances cAMP synthesis by activating adenylyl cyclase, whereas the G
proteins can do so via the
-complex. Hence, we also investigated the role of G
and G
s proteins using the G
s inhibitor NF449, as well as NF023, which inhibits both G
and G
activation with similar potency (15). As shown in Fig. 7A, while PE in the presence of NF023 or G protein antagonist 2A inhibited Ito, treatment with PE in the presence of NF449 prevented the current reduction, suggesting that G
s is required for Ito reduction.
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DISCUSSION |
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1-Adrenergic stimulation reduces Ito via the cAMP/PKA signaling cascade.
Our results show that 1-adrenoceptor stimulation had no effect on Ito after either adenylyl cyclase or PKA inhibition. Moreover, stimulation of either
1-adrenoceptor, adenylyl cyclase or PKA, reduced the Ito amplitude to a similar extent, and the effects of the combined activation of
1-adrenoceptors and adenylyl cyclase or PKA were not additive. These results suggest that the three components act through a common pathway.
One possible limitation of these experiments is that at high concentrations, PE is known to evoke -adrenergic responses. Thus all of these data could be explained if inhibition of Ito were contaminated by
-stimulation. The first experiments conducted with NE plus prazosin excluded this possibility. However, we have confirmed the involvement of
1-adrenoceptors in Ito inhibition by using PE and the more specific
1-agonist methoxamine plus the
-blocker propranolol. Finally, we exclude the involvement of
-adrenoceptors because the
-agonist isoproterenol did not affect Ito.
The most extensively documented biochemical response to 1-adrenoceptor stimulation is G
q-mediated PKC activation. In adult rat ventricles, the novel PKC-
is the major isoform (6), and the PKC-mediated suppression of cardiac K+ currents, including Ito, has been attributed to PKC-
translocation (32, 41). In addition, PKA has also been reported to modulate cardiac Ito (33, 35), to inhibit the K+ A-current in hippocampal neurons and to phosphorylate Kv4.2 (3, 22).
Several studies have demonstrated that in rat, rabbit, and human models, both 1-adrenoceptor stimulation and PKC stimulation reduce Ito amplitude (4, 8, 32, 36, 43). However, none of these authors demonstrated that
1-adrenoceptor stimulation reduced the amplitude of the Ito through PKC activation. In fact, some of them found evidence against this pathway in rat and rabbit (8, 43). Accordingly, in the present study, neither PLC nor PKC inhibition caused a significant reduction in the effect of PE on rat Ito. On the other hand, it has been demonstrated that
1-adrenoceptor stimulation reduces the amplitude of the cardiac Ito through PKC activation in the dog (45). This difference could be due to the different channels that underlie Ito in the different species. Taking into account the molecular basis of Ito in dog (Kv3.4) and in rat and human (Kv1.4 and Kv4.3 in both species), a greater similarity would be expected between humans and rats than between humans and dogs. However, this pathway needs to be explored in experiments in humans.
1-Stimulation increases cAMP levels in localized membrane regions.
Two main questions remain regarding the proposed mechanism of 1-adrenoceptor-induced Ito reduction. The inhibitory response to NE is completely blocked by the
1-adrenoceptor antagonist prazosin. However,
1-stimulation did not increase cAMP levels in the heart (10). Moreover, even if one were to accept the possibility that
1-adrenoceptor activation stimulates cAMP production, prazosin would not block the ability of NE or isoproterenol to stimulate cAMP production via
-adrenoceptors. If Ito is inhibited by a cAMP/PKA-dependent mechanism, why is it that Ito inhibition after
-stimulation is not observed?
One explanation might be that the 1/cAMP response is compartmentalized as has been reported extensively for
-adrenoceptors (for review, Ref. 42). In cardiac myocytes, it is well known that not all cAMP molecules have access to all PKAs and not all PKAs have access to all of their possible substrates. Thus a slight increase in cAMP could lead to an intracellular effect when it is produced at the right place. This means that some
1-adrenoceptor and adenylyl cyclase populations should be in close proximity in a membrane domain that is accessible for Ito channels and for the enzymes regulating them. Thus, when we stimulated
1-adrenoceptors in intact cells, the cAMP levels increase
70%. This increase might seem small compared with the 300400% increase obtained, for example, with
-agonists, glucagon, or forskolin. This is the value obtained in the whole cell, however, and it should be much higher in a given cell domain.
However, when we isolated myocytic membranes, we disrupted possible associations between the 1-adrenoceptor and adenylyl cyclase. Therefore, in isolated membranes,
1-adrenoceptor stimulation did not increase cAMP levels. A recent study (16) demonstrated that the
1-adrenoceptor is found exclusively in the caveolar fraction in cardiac myocytes. Thus caveolae can serve as the scaffolding mechanism for the compartmentalization of the components of this pathway. This is consistent with our results, which show that colchicine prevented the
1-induced Ito reduction because disruption of the cytoskeleton induced caveolae internalization.
To identify the 1-adrenoceptor subtype involved in cAMP production, we selected the
1A-adrenoceptor blocker 5-methylurapidil, the
1B-adrenoceptor blocker chloroethylclonidine, and the
1D-adrenoceptor blocker BMY7378. We used the published dissociation constants (Ki) of each drug for each receptor subtype and the formula R = 100/[1 + (Ki/L)], where R is the occupancy of the receptor (%), and L is the concentration of the antagonist, to calculate drug concentrations that yield an occupancy of
90% of the specific subtype and <5% of the other two subtypes. Our results show that both
1A-adrenoceptor and
1B-adrenoceptor subtypes are coupled to cAMP production, whereas the
1D-adrenoceptor subtype is not. Moreover, selective stimulation of
1A-adrenoceptor or
1B-adrenoceptor increases cAMP production to the maximum level. This fact could mean that the limiting step is not at the receptor level but rather at the level of the specific adenylyl cyclase population.
1-Adrenoceptor stimulation activates G
s.
Several reports have indicated that a receptor can couple to different G proteins. Thus the human substance P receptor directly activates Gq/11, G
s, and G
(40). G
s-coupled receptors such as serotonin, histamine, glucagon, and
2-adrenoceptors also activate G
(27, 47, 48). Moreover, the coupling of these G
s-coupled receptors to G
is functional, given that adenylyl cyclase stimulation by these receptors is increased by treatment with pertussis toxin (47).
Recombinant 1B-adrenoceptors expressed in heterologous systems have been reported to activate G
(5) and G
s (24). Despite the fact that
1-adrenoceptors belong to the larger family of G
q/11 protein-coupled receptors, native
1B-adrenoceptors may also couple to G
in rat aortic smooth muscle (20), although the functional relevance of this coupling is not yet clear.
In this report, we first describe that in isolated rat ventricular myocytes, the 1-adrenoceptor-induced reduction of Ito is not dependent on G
q activation, because the blockade of G
q activation did not prevent the PE-induced effect. However, the blockade of G
s activation completely suppressed the current inhibition induced by
1-adrenoceptors. Second, we show that
1-adrenoceptors and G
s proteins are associated in ventricular myocytes, that this association increases after receptor stimulation, and that the coupling between the receptor and the G
s is functional because it is fully displaced by GTP
S and prevented by NF449.
In summary, the present study provides evidence that 1-adrenoceptor stimulation couples the receptor to a G
s protein. This coupling to G
s is functional and activates the cAMP/PKA signaling cascade, which leads to Ito phosphorylation and reduction. This appears to be the first example, in a physiological preparation, in which
1-adrenoceptors are linked to the stimulation of cAMP production and subsequent activation of a PKA-dependent response.
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GRANTS |
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ACKNOWLEDGMENTS |
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Present address of M. Gallego: Huntsman Cancer Institute, University of Utah, 2000 Circle of Hope Dr., Salt Lake City, UT 84112-5550.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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