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Am J Physiol Heart Circ Physiol 284: H619-H625, 2003. First published October 10, 2002; doi:10.1152/ajpheart.00110.2002
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Vol. 284, Issue 2, H619-H625, February 2003

Interaction between ATP and catecholamines in stimulation of platelet aggregation

Alex V. Birk1,2, Endri Leno3, Hugh D. Robertson2, Victoria M. Bolotina3, and Hazel H. Szeto1

Departments of 1 Pharmacology and 2 Biochemistry, Weill Medical College of Cornell University, New York, New York 10021; and 3 Department of Physiology, Boston University School of Medicine, Boston, Massachusetts 02118-2393


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Platelets, on activation by endothelial damage, release ADP, ATP, serotonin, epinephrine, and norepinephrine. Although ATP is known to augment the action of norepinephrine in cardiovascular and endocrine systems, the possible interaction between ATP and catecholamines in regulation of platelet reactivity has not been reported. The addition of ATP (1-5 µM) to human platelet-rich plasma did not induce platelet aggregation; however, it selectively augmented the aggregatory response to norepinephrine and epinephrine, but not to serotonin. This potentiating action of ATP was dose dependent and was not due to contamination by, or hydrolysis to, ADP. The action of ATP was blocked by 10 µM of adenosine 3'-phosphate 5'-phosphosulfate, a selective P2Y1 receptor antagonist. ATP alone did not cause release of intracellular Ca2+, but produced a significant Ca2+ response in the presence of norepinephrine. In contrast, the P2X1 receptor agonists P1,P6-diadenosine-5' hexophosphate and alpha ,beta -methylene-ATP had no effect on norepinephrine-induced platelet aggregation even when added at 100 µM. This synergistic interaction between ATP and norepinephrine in stimulating platelet aggregation may have significant clinical implications and suggests a prothrombotic role for ATP in stress.

norepinephrine; synergism


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PLATELETS PLAY A MAJOR ROLE in the maintenance of endothelial integrity and hemostasis. On activation by endothelial damage, platelets undergo morphological changes and release the contents of their dense granules, including ADP, ATP, serotonin (5-HT), epinephrine (Epi), and norepinephrine (NE). Interactions among these substances play a role in regulating further recruitment of platelets to the site of vascular injury.

ATP and ADP have been reported to produce opposing effects on platelet aggregation. ADP induces platelet aggregation via two distinct G protein-coupled receptors, P2Y1 and P2Y12 (or P2YAC) (20). P2Y1 is coupled to Gq and activation of PLC-intracellular Ca2+ pathway, and P2Y12 is coupled to Gi and inhibition of adenylyl cyclase (20). Although ATP does not appear to have any proaggregatory action, it has been postulated to be an antiaggregatory agent based on the finding that ATP inhibits ADP-induced platelet aggregation. However, the concentration of ATP required for this inhibitory action is 50- to 100-fold in excess of ADP concentrations (32). Although ATP has been considered an antagonist at platelet P2Y1 and P2Y12 receptors (23, 32, 40), this antagonistic action of ATP may be questioned given the high concentration required and because ATP has been reported to be an agonist in most of the studies (12, 15, 37, 49) that used recombinant or native P2Y1 receptors. Moreover, recent studies (41, 42) have shown that low concentrations of ATP significantly enhanced the aggregation response to the thromboxane A2 analog U-44619 and collagen. Thus the physiological role of ATP on platelet aggregation remains controversial.

The catecholamines are not considered to be true mediators of platelet aggregation. 5-HT alone, acting at 5-HT2 receptors, does not induce aggregation of human platelets, although it can potentiate platelet aggregation induced by NE (38). Both Epi and NE, acting at alpha 2A-receptors, have been shown to induce aggregation and to potentiate the aggregatory response to ADP, thrombin, and thromboxane (30). The aggregatory response to NE and Epi is biphasic with a small initial response, followed by a much stronger secondary response (30, 33, 43). It has been suggested that the secondary aggregation phase of Epi is mediated via the release of ADP (10). However, creatine phosphate (CP) plus creatine phosphokinase (CPK) (CP/CPK) failed to inhibit the secondary aggregation induced by Epi (16, 22). Because CP/CPK converts ADP to ATP, that finding suggests a role for ATP in the aggregatory action of Epi.

Interactions between NE and ATP have been reported in several physiological systems. NE and ATP are coreleased from sympathetic nerve terminals (28) and synergistic interactions between them have been demonstrated in the mouse vas deferens (17, 51) and cardiac myocytes (8). Furthermore, ATP and NE have been shown to synergize in the release of vasopressin and oxytocin from the hypothalamus (21). Although ATP is coreleased with NE, Epi, and 5-HT from platelets (39, 45), it is not known whether ATP might potentiate the action of catecholamines in regulation of platelet reactivity. Thus the goals of the present study were 1) to determine interactions between ATP and NE, Epi, or 5-HT in platelet aggregation, and 2) to investigate the possible mechanism(s) involved in these interactions.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals. ADP, ATP, 5-HT, Epi, NE, alpha ,beta -methylene-ATP (alpha ,beta -Me-ATP), P1,P6-diadenosine-5' hexaphosphate (Ap6A), adenosine 3'-phosphate 5'-phosphosulfate sodium salt (A3P5PS), and fura 2-AM were all purchased from Sigma (St. Louis, MO).

HPLC. The purity of the ATP was confirmed with the use of HPLC. ATP was eluted on a C18 column using a linear gradient with buffer A (10 mM tetrabutyl ammonium hydroxide, 10 mM KH2PO4, and 0.25% MeOH, pH 7.0) and buffer B (2.5 mM tetrabutyl ammonium hydroxide, 100 mM KH2PO4, and 30% MeOH, pH 5.5). AMP, ADP, and ATP were eluted at 11.1, 19.0, and 34.1 min, respectively.

Preparation of human platelet-rich plasma. The protocol for use of human subjects was approved by the Weill Medical College of Cornell University Institutional Review Board. Blood was collected from healthy volunteers who had not taken any medications for at least 10 days before the study. Blood was collected by venipuncture into plastic tubes containing heparin at a final concentration of 5 U/ml. Platelet-rich plasma (PRP) was obtained by centrifugation of the blood sample for 15 min at 200 g and for another 10 min at 120 g.

Preparation of human platelet-poor plasma. Blood was collected as described above. It was centrifuged at 1,900 g twice for 15 min each time to remove the blood cells. In addition, the plasma was centrifuged once more at 14,000 g for 5 min just before use.

Determination of ATP hydrolysis in human PRP. A trace of [gamma -32P]ATP mixed together with ATP in Tyrode buffer composed of (in mM) 145 NaCl, 4 KCl, 1 MgCl2, 1 CaCl2, 0.5 NaH2PO4, 6 glucose, and 10 HEPES Cl (pH 7.4) was added alone or with NE to 300 µl PRP to a final concentration of 5 µM ATP and 10 µM NE. After preincubation for 0, 1, 2, or 4 min at 37°C, the reaction was stopped with 30 µl of 500 mM EDTA (pH 8.5). ATP alone in buffer or ATP pretreated with alkaline phosphatase (Promega; Madison, WI) was used as control and Pi marker, respectively. The reaction products ([gamma -32P]ATP, 32PPi, and 32Pi) were fractionated by paper electrophoresis on Whatman 3MM paper in a buffer containing 5% acetic acid and 0.5% pyridine at pH 3.5 (4) and quantified with phosphorimaging. The limit of detection of 32P-labeled compounds ([gamma -32P]ATP, 32PPi, and 32Pi) in our assay was 50 nM. The formation of 32PPi or 32Pi from [gamma -32P]ATP suggests that ATP was hydrolyzed to AMP or ADP, respectively.

Platelet aggregation studies. Platelet aggregation was determined with 300 µl of PRP containing 250,000 platelets/µl (Chronolog Lumi-Aggregometer; Havertown, PA). Platelet-poor plasma (PPP) was used as a blank for 100% aggregation reference.

Ca2+ measurements. Changes in intracellular Ca2+ in platelets were monitored using standard fura 2 technique, as previously described (46). PRP (500 µl) was loaded with 2.5 µM fura 2-AM and incubated at 37°C for 15 min. The loaded PRP was diluted with standard citrate buffer [137 mM NaCl, 2.7 mM KCl, 0.98 mM MgCl2, 3.3 mM NaH2PO4, 5.5 mM glucose, 100 µM aspirin, 5% citrate (wt/vol), and 10 mM HEPES, pH 7.4] and centrifuged at 160 g for 5 min at room temperature. Platelets were resuspended in the same buffer without citrate to the final concentration of approx 108 cells/ml. To measure Ca2+ release from the stores, ATP and NE were applied to citrate buffer-washed platelets in the presence of 100 µM EGTA. Fluorescence (F) measurements were carried out at 37°C with the use of a spectrofluorimeter (Hitachi F-4500) with the excitation wavelength alternating between 340 and 380 nm every 0.5 s and the emission wavelength set at 510 nm. Intracellular Ca2+ was monitored using the F340-to-F380 ratio (F340/F380), and changes in Ca2+ were measured as the difference between the peak Ca2+ release and the basal level and were expressed as Delta F340/F380. Data collection, ratio calculation, and analysis were performed with the Hitachi software.

Data analysis. All experiments for platelet aggregation, nucleotides hydrolysis, and Ca2+ measurements were carried out in duplicates with three different subjects. All data are presented as means ± SE.

The area under the platelet aggregation curve was determined with the use of compensating polar planimeter (Keuffel and Esser; Morristown, NJ). The rate of rise of the aggregation was determined as the slope of the aggregation curve in a first 30 s. Response to the various concentrations of ATP (0-5 µM) in the presence 10 µM NE was expressed as the percent change compared with NE alone.

The rate of PPi and Pi formation from ATP in PRP were determined by linear regression analysis. The effect of NE on the rate of PPi and Pi formation was ascertained by one-way ANOVA, with Newman-Keuls test for post hoc comparison. Differences were considered to be significant when P < 0.05.

The Ca2+ responses were quantified using the Delta F340/F380 of peak-to-basal noise. The Ca2+ responses were further analyzed by one-way ANOVA, with Newman-Keuls test for post hoc comparison between ATP alone and in combination with NE. Differences were considered to be significant when P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Although 5 µM ATP alone did not induce platelet aggregation when added to human PRP, it significantly potentiated the aggregatory response to NE (10 µM) (Fig. 1 and 2). NE alone, even at a dose of 10 µM, produced only a small aggregatory response (Fig. 1). The addition of ATP (0-5 µM) in the presence NE (10 µM) significantly increased the magnitude of the aggregation (Fig. 2A) and the initial rate of aggregation (Fig. 2B) in a dose-dependent manner. ATP also potentiated the aggregatory response to Epi (data not shown). However, ATP (5 µM) failed to cause any aggregation in the presence of 10 µM 5-HT (Fig. 1).


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  		Fig. 1.   Effect of ATP on norepinephrine (NE)- and serotonin (5-HT)-induced platelet aggregation. Representative recordings of platelet aggregation in the presence of ATP, NE, and 5-HT are plotted against time. Although ATP (5 µM) did not induce platelet aggregation, it potentiated platelet aggregation induced by NE (10 µM) but was ineffective when added together with 5-HT. All experiments were carried out for 4 min. Arrows indicate the addition of various compounds. This figure is a representative recording from three different subjects showing similar results.



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  		Fig. 2.   ATP potentiates NE-induced aggregation in a dose-dependent manner. A: ATP (1-5 µM) potentiated NE (10 µM)-induced platelet aggregation as determined by the area under the platelet aggregation curve over 4 min. B: initial rate of aggregation (first 30 s of aggregatory reaction). Data are presented as means ± SE (n = 3).

To determine that ATP-mediated potentiation of NE-induced platelet aggregation was not due to ADP contamination in the sample, the purity of the ATP used in these studies was confirmed by HPLC. The chromatogram of the ATP sample is shown in Fig. 3A. The level of ADP contamination was estimated to be <0.5%. Thus the concentration of ADP in the 5 µM ATP sample should be <25 nM. The effect of ATP was also not due to hydrolysis of ATP to ADP in PRP. The hydrolysis of ATP was examined with the use of [gamma -32P]ATP (Fig. 3B). The major product found was 32PPi, with a rate of formation of 340.8 ± 24.9 pmol · min-1 · ml-1, which was significantly reduced by the addition of 10 µM NE (223.0 ± 9.9 pmol · min-1 · ml-1) (P < 0.05). Although the formation of Pi, corresponding to production of ADP, was detected, the rate of Pi formation was only 109 ± 7.4 pmol · min-1 · ml-1 and was not changed in the presence of 10 µM NE (115 ± 4.4 pmol · min-1 · ml-1). Figure 3C illustrates that the addition of ADP at a concentration (0.1 µM) significantly higher than contaminating levels or levels formed as a result of hydrolysis of ATP in PRP did not potentiate NE-induced platelet aggregation.


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  		Fig. 3.   Purity of ATP and its metabolism in human platelet-rich plasma. A: HPLC chromatogram of ATP sample used for platelet aggregation and metabolism studies. The retention times for ADP and ATP were 19 and 35 min, respectively. B: NE did not affect ADP formation but inhibited AMP formation in human PRP. The rate of formation of PPi and Pi from 5 µM [gamma -32P]ATP were measured in the absence or presence of 10 µM NE. *P < 0.05 comparing PPi formation in the absence and presence of NE. C: ADP (0.1 µM) had no effect on platelet aggregation induced by NE (10 µM). Representative recordings of platelet aggregation are plotted against time. All experiments were carried out for 4 min. Arrows indicate the addition of various compounds. This figure is a representative recording from three different subjects showing similar results. AU, arbitrary units.

ATP is known to be an agonist at both P2Y1 and P2X1 receptors. To determine the involvement of P2Y1 receptors in the ATP/NE interaction, the selective P2Y1 antagonist A3P5PS was used. The interaction between ATP and NE was abolished by 10 µM A3P5PS (Fig. 4). In contrast, even very high concentrations (100 µM) of the selective P2X1 receptor agonists Ap6A and alpha ,beta -Me-ATP did not potentiate the platelet aggregation response to NE (Fig. 4).


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  		Fig. 4.   Role of P2Y1 and P2X1 receptors in ATP-potentiating NE-induced platelet aggregation. Representative recordings of platelet aggregation are plotted against time. The ability of ATP (2.5 µM) to potentiate platelet aggregation induced by NE (10 µM) is blocked by the P2Y1 receptor antagonist adenosine 3'-phosphate 5'-phosphosulfate sodium salt (A3P5PS) (10 µM). Neither alpha ,beta -methylene (Me)-ATP (100 µM) nor P1,P6-diadenosine-5' hexaphosphate (Ap6A) (100 µM), both selective P2X1 agonists, potentiated platelet aggregation induced by NE (10 µM). All experiments were carried out for 4 min. Arrows indicate the addition of various compounds. This figure is a representative recording from three different subjects showing similar results.

Intracellular Ca2+ in washed platelets was measured to determine whether intracellular Ca2+ release is involved in ATP-NE interaction. To eliminate the potential involvement of P2X1 receptors and Ca2+ influx, all experiments were done in Ca2+-free medium. Neither ATP (100 µM) nor NE (10 µM) alone produced any significant change in intracellular Ca2+. The simultaneous addition of both ATP (100 µM) and NE (10 µM) together produced a significant and stable increase in Ca2+ release (Fig. 5).


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  		Fig. 5.   Effect of ADP, ATP, and NE on intracellular Ca2+ release. Changes in intracellular Ca2+ were measured in the absence of extracellular Ca2+ to avoid Ca2+ influx. A: representative recordings of changes in intracellular Ca2+ in response to ATP (100 µM) and NE (10 µM) added separately or together (right) compared with response to ADP (10 µM) (left). This figure is a representative recording from three different subjects showing similar results. B: summary data showing the changes in intracellular Ca2+ [expressed as the difference in fluorescence ratios at 340 and 380 nm (Delta F340/F380)] caused by separate applications of ATP (100 µM), ADP (10 µM), and NE (10 µM) alone and ATP and NE together. *P < 0.05 compared with NE alone.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present findings show that extracellular ATP can potentiate NE-induced platelet aggregation. ATP alone had no effect on platelet aggregation. However, the addition of ATP together with NE produced a much larger aggregatory response than can be generated by NE alone. ATP also potentiated the aggregatory response to Epi. This potentiating effect of ATP was dose dependent and was observed with ATP doses 10-100 times lower than those needed for inhibition of ADP-induced platelet aggregation (32). In contrast to the interaction seen between ATP and NE/Epi, the addition of ATP and 5-HT did not elicit aggregatory response, suggesting that the interaction between ATP and NE/Epi is rather specific.

The interaction between ATP and NE appears to be due to a direct action of ATP and not as a result of contamination by ADP in the source material. The purity of our ATP sample was confirmed by HPLC and the results showed <0.5% ADP contamination. Moreover, the rate of ADP formation from ATP in PRP is too slow to account for the rapid aggregatory response observed on simultaneous addition of ATP and NE. In addition, 0.1 µM ADP, a concentration four times higher than the contaminating level of ADP in our 5 µM ATP sample and two times higher than could be produced during ATP metabolism in PRP, did not potentiate platelet aggregation in the presence of NE.

Three purinergic receptors have been identified on platelets: P2X1, P2Y1, and P2Y12. ATP is known to act as an agonist at the P2X1 receptor, which is an ATP-gated Ca2+ channel (7). Patch-clamp studies (25) with human platelets showed that application of ATP or its unhydrolyzable analog ATPgamma S evoked a transient inward current. These authors subsequently reported that the Ca2+ influx in response to ADP was actually due to contamination of the commercial ADP sample by ATP, and purified ADP was not an agonist at P2X1 receptors (26). Furthermore, it was also reported that ATP acting via the P2X1 receptor could induce transient platelet shape change (35). However, the selective P2X1 receptor agonists alpha ,beta -Me-ATP and Ap6A (29) failed to potentiate NE-induced platelet aggregation in our study. In contrast, the interaction between ATP and NE was blocked by A3P5PS, a selective P2Y1 antagonist, suggesting that P2Y1 receptors are involved in ATP-NE interaction. A3P5PS is >10 times more selective for P2Y1 than for P2Y12, with IC50 being 8 µM for inhibition of P2Y1-mediated Ca2+ mobilization compared with IC50 of >100 µM for P2Y12-mediated inhibition of adenylyl cyclase (44). At the concentration used in our study (10 µM), A3P5PS should be quite selective for the P2Y1 receptor. Interestingly, this dose is 10 to 100 times lower than the one commonly used for blocking P2Y1 receptors in previous platelet studies (19, 36). Although some previous studies have cast doubt on the ability of ATP to activate P2Y1 receptors and raised questions regarding the purity and possible degradation of the ATP (14, 23), it was recently reported that ATP and ADP are both agonists at the recombinant P2Y1 receptor (11). These discrepancies may be due to different levels of receptor expression and the lower efficacy of ATP at the P2Y1 receptor (31), possibly acting as a partial agonist. In addition, ATP is unlikely to be an antagonist at P2Y1 receptors because its concentration over ADP in our aggregation studies was high enough (at least 100-fold) to block action of ADP at P2Y1 receptors, thus preventing aggregation. Because our data clearly demonstrated synergism between ATP and NE, it is possible that NE sensitizes P2Y1 receptor to the action of its weak agonist, ATP.

We further investigated the role of P2Y1 receptor by measuring intracellular Ca2+ release in the absence of extracellular Ca2+, thereby eliminating possible contribution of the P2X1 receptor to intracellular Ca2+ changes and minimizing extracellular ATP hydrolysis. We observed that ADP produced a significant increase in intracellular Ca2+, suggesting the presence of functional P2Y1 receptors. NE potentiated ADP-induced intracellular Ca2+ release suggesting synergistic interaction between P2Y1 and alpha 2A-receptors. However, ATP, even at the concentrations 10 times larger than ADP, did not produce any detectable Ca2+ response, suggesting that ATP alone has low efficacy at the platelet P2Y1 receptor. To our surprise, although NE also did not stimulate intracellular Ca2+ release, the addition of both ATP and NE together induced comparable increase in Ca2+ release as ADP alone, suggesting that ATP-NE interaction could be at the level of intracellular Ca2+ release pathways. In agreement with our aggregation data, these Ca2+ data also suggest that NE might sensitize the P2Y1 receptor to the action of ATP.

The mechanism behind this observed synergism between ATP and NE is not clear. NE acts on alpha 2A-receptors that are coupled to Gi and inhibition of adenylyl cyclase, whereas P2Y1 receptors are coupled to Gq and activation of PLC. In addition, the recombinant alpha 2A-receptor has been reported to couple to activation of PLC via the Gi-associated beta gamma -subunits (1, 5, 9). However, this action of the recombinant alpha 2A-receptor in Chinese hamster ovary cells was blocked by treatment with apyrase, revealing that endogenous ATP acting at P2Y receptors may direct alpha 2A-receptor coupling to mobilization of intracellular Ca2+ (1, 9). Furthermore, it was subsequently shown that preactivation of PLC via Gq-coupled receptor is required for activation of PLC-intracellular Ca2+ release pathway by Gi-associated beta gamma -subunits (6), suggesting that activation of endogenous Gq coupled receptors is likely to stimulate alpha 2A-receptor coupling to PLC pathway. In addition to ATP stimulating the action of NE, NE was shown to sensitize the P2Y1 receptor to the action of ATP by activating the transfer of Gi-associated beta gamma -subunit to preactivated Galpha q and enhancing the exchange of GDP/GTP and stimulation of PLC (34). Thus interaction between ATP and NE could be bidirectional, resulting in potentiation of each other's action.

The rapid hydrolysis of ATP to AMP in plasma is consistent with previous reports (2, 3, 27) on soluble phosphodiesterase-like activity in plasma. Formation of ADP in PRP was slow and is likely to be due to ecto-ATPase or ectokinase on platelets. The phosphodiesterase-like activity in plasma might explain the absence of aggregatory response in the presence of ATP alone. The formation of AMP and its rapid hydrolysis in plasma (approx 5,000 pmol · min-1 · ml-1) (data not shown) would generate adenosine, a potent inhibitor of platelet aggregation. The inhibition of ATP hydrolysis to AMP by NE would further contribute to the potentiation of NE-induced platelet aggregation by ATP. Because ATP hydrolysis in PPP was not affected by NE (data not shown), we hypothesize that NE promoted the release of exophosphodiesterase inhibitory factor(s) from platelets.

Our studies did not employ potato apyrase for platelets preparation. Potato apyrase contains ATPase activity, which could metabolize ATP to ADP, thus complicating data interpretation. In addition to ATPase and ADPase activities, we have found that potato apyrase (grade VII) (Sigma), can also rapidly hydrolyze both paranitrophenol phosphate and paranitrophenol-5'-thymidine monophosphate (data not shown), specific substrates for alkaline phosphatase and phosphodiesterase, respectively. Finally, because potato apyrase is only a partially purified extract from potato, it is likely to contain other enzymatic activities (such as proteases) that could have stimulatory or inhibitory effects on platelet aggregation.

The results from this study illustrate the complex interactions that exist among the various substances that are released from the dense granules of activated platelets. It is apparent that synergistic interactions among these mediators can profoundly enhance platelet aggregation, and in vitro studies using a single agent do not necessarily reveal the complexity under endogenous conditions. The interaction between ATP and NE/Epi on platelet aggregation may have significant clinical implications and suggests a prothrombotic role for ATP in stress. In addition to release from platelets, Epi is released from the adrenal medulla during stress, and NE is released together with ATP from sympathetic nerve terminals (24, 28, 47, 48, 50). Thus, under conditions of stress, ATP and ADP are always acting in the presence of catecholamines. Elevations in Epi and NE concentrations in blood during stress are thought to promote thrombosis even without significant endothelial damage because of their vasoconstrictive properties and direct action on platelets via alpha 2A receptors. Increase in plasma NE by direct NE infusion or by stress paradigms has been reported to result in intravascular platelet aggregation in animals (13) and humans (18). During cardiac ischemia, ATP is coreleased with NE from cardiac sympathetic nerves into the coronary circulation. This combination is likely to result in intravascular platelet aggregation, which would further aggravate cardiac ischemia. This constant association between ATP and catecholamines creates an environment where any antithrombotic action of ATP is likely to be overridden.


    ACKNOWLEDGEMENTS

We thank Drs. Aaron J. Marcus and M. Johan Broekman for invaluable advice and support throughout these studies. We also thank Dr. Joel D. Pardee and Dr. Susanna Hourani for helpful discussions and Dr. Jochen Buck for help with the HPLC.


    FOOTNOTES

This work was supported by National Institutes of Health Grants DA-02475 (to A. V. Birk), DA-08924 (to H. H. Szeto), HL-54150 (to V. M. Bolotina), and DK-56424 (to H. D. Robertson).

Address for reprint requests and other correspondence: H. H. Szeto, Dept. of Pharmacology, Weill Medical College of Cornell Univ., 1300 York Ave., Rm. LC405, New York, NY 10021 (E-mail: hhszeto{at}med.cornell.edu).

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.

First published 10 October 2002;10.1152/ajpheart.00110.2002

Received 8 February 2002; accepted in final form 8 October 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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