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CARDIOVASCULAR
Departments of Pharmacology (T.M., U.S., R.L.), Anesthesiology (P.M.H.), and Physiology and Biophysics (A.C.R., R.B.S.), and Division of Hematology and Medical Oncology, Departments of Medicine (M.J.B., A.J.M.) and Pathology (A.J.M.), Weill Medical College of Cornell University, New York, New York; and Division of Hematology and Medical Oncology, Department of Medicine (M.J.B., A.J.M.), VA New York Harbor Health Care System, New York, New York
Received November 22, 2004; accepted January 11, 2005.
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Abstract |
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-conotoxin-inhibitable release of both norepinephrine and ATP. Inhibition of E-NTPDase1/CD39 with ARL67156markedly potentiated ATP release, demonstrating that E-NTPDase1/CD39 is a major determinant of ATP availability at sympathetic nerve terminals. Notably, inhibition of E-NTPDase1/CD39 enhanced both ATP and NE exocytosis, whereas administration of soluble CD39 reduced both ATP and NE exocytosis. The strong correlation between ATP and norepinephrine release was abolished in the presence of the purinergic P2X receptor (P2XR) antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). We conclude that released ATP governs norepinephrine exocytosis by activating presynaptic P2XR and that this action is controlled by neuronal E-NTPDase1/CD39. Clinically, excessive norepinephrine release is a major cause of arrhythmic and coronary vascular dysfunction during myocardial ischemia. By curtailing NE release, in addition to its effects as an antithrombotic agent, soluble CD39 may constitute a novel therapeutic approach to ischemic complications in the myocardium.
; Sneddon et al., 1999). Whether NE and ATP are released from the same site or from different varicosities (Driessen et al., 1993; Westfall et al., 1996; Bobalova and Mutafova-Yambolieva, 2001; Stjarne, 2001; Brock and Tan, 2004), ATP is likely to modulate NE release in either case (von Kugelgen et al., 1999; Sperlágh et al., 2000; Sesti et al., 2002). Indeed, once released, neurotransmitter ATP promotes NE release by activating presynaptic P2X purinoceptors (P2XRs) (Boehm, 1999; Sperlágh et al., 2000; Sesti et al., 2002; Queiroz et al., 2003).
Two different and independent mechanisms terminate the effects of the two transmitters: NE is removed from the synaptic cleft primarily by reuptake into the nerve terminals by a specific transporter (Amara and Kuhar, 1993), whereas ATP is metabolized by nucleotidases (Zimmermann and Braun, 1999; Westfall et al., 2002). Using sympathetic nerve endings isolated from guinea pig heart (i.e., cardiac synaptosomes), we previously demonstrated that activation of ATP-gated ionotropic presynaptic P2XRs promoted NE exocytosis (Sesti et al., 2002). This effect was increased by inhibition of endogenous ectonucleotidase (E-NTPDase1) and diminished by the addition of soluble CD39 (solCD39), a recombinant form of human E-NTPDase1 (Sesti et al., 2002; Marcus et al., 2003). Furthermore, depolarization of synaptosomes with K+ evoked NE exocytosis, which was also potentiated by inhibition of E-NTPDase1 and attenuated by administration of solCD39.
These findings suggest that ATP released by depolarization of sympathetic terminals enhances NE exocytosis and that E-NTPDase1 plays an important role in adrenergic neurotransmission in the heart. This notion was based on results obtained in synaptosomes isolated from guinea pig heart, but it required verification and amplification in an intact tissue preparation. Accordingly, we have now studied the localization of CD39 in human and porcine heart. In addition, we studied the role of E-NTPDase1 in exocytosis of NE and ATP in porcine heart, the physiology of which more closely resembles the human heart (Appel et al., 2001). Our findings have now directly demonstrated the presence of E-NTPDase1/CD39 in cardiac neurons and indicate that, by terminating the action of transmitter ATP, E-NTPDase1/CD39 negatively modulates NE exocytosis in the heart.
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Materials and Methods |
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1 cm3 was removed from the atriotomy site and rapidly transported to the laboratory in ice-cold oxygenated Krebs-Henseleit solution (KHS) composed of the following: 118.2 mM NaCl, 4.83 mM KCl, 2.5 mM CaCl2, 2.37 mM MgSO4, 1.0 mM KH2PO4, 25 mM NaHCO3, and 11.1 mM glucose. Porcine. Under a protocol approved by the Institutional Animal Care and Use Committee, tissue harvested from eight female Sinclair pigs weighing approximately 30 kg were used for the study. After sedation with intramuscular tiletamine/zolazepam (2.2 mg/kg) and xylazine (2.2 mg/kg), animals were deeply anesthetized with isoflurane in oxygen via facemask, and the trachea was intubated. The lungs were subsequently ventilated with isoflurane (1.3% endtidal) and 100% oxygen. The chest was opened via median sternotomy, the pericardium was opened widely, and the heart was surrounded with ice. With the animal deeply anesthetized, the heart was arrested with an iced potassium chloride solution, rapidly removed, and placed in iced saline solution. Approximately half the right atrial appendage and 50 g of the left ventricle free wall were removed and transferred to oxygenated buffer for subsequent preparation.
Incubation Conditions
Immediately after dissection, specimens of porcine atrial and ventricular tissue were transported to the laboratory in ice-cold oxygenated KHS. After the removal of fat and connective tissue, samples were divided into several fragments (each weighing 17.3 ± 0.6 mg wet weight, measured at the end of incubation). Each fragment was incubated for 20 min at 37.5°C in 2 ml of KHS gassed with 95% O2 and 5% CO2 (pO2 of 550 mm Hg, pH 7.4) containing the monoamine oxidase inhibitor pargyline (1 mM). After this 20-min incubation period, fragments were incubated for an additional 15 min in oxygenated KHS in the absence or presence of one or more pharmacological agents. These included solCD39, a recombinant, soluble form of human E-NTPDase1/CD39 (Gayle et al., 1998), or the E-NTPDase1 inhibitor ARL67156(Crack et al., 1995), the P2XR antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (Lambrecht, 2000), and the N-type Ca2+-channel antagonist -conotoxin (-CTX) (Sher et al., 1991).
NE Exocytosis
At the conclusion of the 15-min incubation with pharmacological agents, fragments of porcine atrial and ventricular tissue were incubated for 5 min in either normal (5.83 mM K+) or depolarizing (50 and 100 mM K+) KHS containing 1 mM pargyline and tropolone (catechol-O-methyltransferase inhibitor), and 1 µM each of atropine (muscarinic antagonist), desipramine (NE transporter inhibitor), and yohimbine (
2-adrenoceptor antagonist). When studied, pharmacological agents were present in the depolarizing solution.
NE Assay
Incubating solutions were assayed for NE using high-pressure liquid chromatography with electrochemical detection (Maruyama et al., 2000). Perchloric acid and EDTA were added to samples to achieve final concentrations of 0.01 N and 0.025%, respectively. After a short period of storage at –70°C, the samples were thawed. The NE present in the medium was adsorbed on acid-washed alumina, adjusted to pH 8.6 with Tris-2% EDTA buffer, and then extracted into 150 µl of 0.1 N perchloric acid. Aliquots were injected onto a 3-µm octadecylsilane reverse phase column (3.2 x 100 mm; BAS Bioanalytical Systems, West Lafayette, IN) with an applied potential of 0.65 V. The mobile phase consisted of 7.5 mM monochloroacetic acid, 0.5 mM sodium EDTA, 0.5 mM sodium octylsulfate, and 1.5% acetonitrile at pH 3.0. The flow rate was 1.0 ml/min. Dihydroxybenzylamine was added to each sample as an internal standard before alumina extraction and was used for calculation of recovery during the extraction procedure.
ATP Assay
ATP levels were measured using a firefly luciferin-luciferase assay-based commercial kit (ATP bioluminescence assay kit HS II; Roche Diagnostics, Indianapolis, IN) (Sesti et al., 2003). Samples (50 µl) of incubating solution were dispensed into appropriate test tubes, placed in a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA), and processed by autoinjection of 50 µl of luciferin-luciferase reagent. ATP concentrations were calculated from a calibration curve constructed the same day using ATP standards provided in the kit. The amount of ATP was expressed as nanomoles per gram of tissue.
Immunohistochemistry
Tissue Preparation. Immediately after dissection, specimens of human and porcine atrial tissue were transported to the laboratory in ice-cold oxygenated KHS and immersed in a 4% paraformaldehyde solution, pH 7.4, for 1 h for fixation before rinsing and storing in 30% sucrose for 3 h to cryoprotect. The tissue was then embedded in tissue-freezing medium (Electron Microscopy Sciences, Fort Washington, PA) and snap-frozen in liquid nitrogen. Using a Bright cryostat (model OTF), frozen sections of 10 µm were prepared, collected onto Fisher Superfrost Plus slides, and stored at –80°C until ready for immunohistochemistry.
Immunohistochemical Staining. Slides containing frozen tissue sections were washed for 5 min in PBS. The sections were then permeabilized for 10 min at 37°C with 0.3% Triton X-100 dissolved in either 4% fetal bovine serum (human tissue) or donkey serum (porcine tissue). After washing the sections with PBS, either 10% fetal bovine serum or donkey serum was applied to the sections for 1 h at 37°C to block nonspecific binding before adding antibodies. Then primary antibody (1° Ab) was applied to the sections for 2 h at 37°C, followed by three washes in PBS. Next, sections were exposed to secondary antibody (2° Ab) for 1 h at 37°C. Sections were then washed with PBS as described above, followed by fixation for 3 min with 4% paraformaldehyde solution. After washing with PBS, sections were mounted in Vectashield anti-fading solution (Vector Laboratories, Burlingame, CA).
In human sections that were probed with only a mouse monoclonal human anti-CD39 antibody (1:100), the corresponding 2° Ab was Alexa Fluor 488 goat anti-mouse IgG (1:400). For colocalization studies, both a rabbit anti-synapsin I antibody, a specific label of neurons (De Camilli et al., 1983), and a mouse monoclonal human anti-CD39 antibody were applied to human heart sections at a dilution of 1:100. For these sections, the 2° Abs used were Alexa Fluor 594 donkey anti-rabbit IgG (red) (1:1200) and Alexa Fluor 488 donkey anti-mouse IgG (green) (1:600).
Porcine heart sections that were colabeled with the mouse monoclonal human anti-CD39 antibody (1:100) and goat polyclonal anti-synapsin Ia/b antibody (1:400) were subsequently stained with the following 2° Abs: Alexa Fluor 488 donkey anti-mouse IgG (green) (1:600) and Alexa Fluor 594 donkey anti-goat IgG (red) (1:1200).
Tissue sections were examined with an inverted epifluorescent microscope (Nikon Diaphot; Morrell Instrument Co., Melville, NY) interfaced to a frame-transfer type cooled charge-coupled device (Roper Scientific, Duluth, GA) and processed with MetaFluor/MetaMorph software (Universal Imaging Corporation, Downingtown, PA). Digital images were imported into Adobe Photoshop (7.0) for minimal processing.
Drugs
SolCD39 was a generous gift from Drs. C. R. Maliszewski and R. B. Gayle III (Amgen/Immunex, Seattle, WA). ARL67156was purchased from A. G. Scientific, Inc. (San Diego, CA). Donkey serum was purchased from Abcam (Cambridge, MA). Mouse monoclonal anti-human CD39 antibody BU61 was purchased from Accurate Chemical & Scientific (Westbury, NY). Goat polyclonal anti-synapsin Ia/b antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All other antibodies were purchased from Molecular Probes (Eugene, OR). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
Statistics
Values are expressed as mean ± S.E.M. Analysis by one-way ANOVA was followed by post hoc testing (Dunnett's test). A value of P < 0.05 was considered statistically significant.
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Results |
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) and the anti-CD39 antibody. We found that CD39 and synapsin were colocalized in cardiac nerves, as shown in Fig. 1, B–D. Given the close similarity of human and porcine cardiac tissues (Appel et al., 2001), and because of limited availability of human heart specimens, we next extended these studies to porcine atrial tissue. As in the human heart, CD39 and synapsin colocalized in porcine cardiac nerves (Fig. 1, E–G).
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We next studied the role of E-NTPDase1/CD39 in the exocytosis of NE and ATP. As shown in Fig. 2, K+-induced depolarization of porcine atrial (A) and ventricular (B) tissue elicited release of NE and ATP. In response to 50 and 100 mM K+, NE release increased 2- and 8- to 9-fold above control values, respectively, in both atria and ventricles. With 50 and 100 mM K+, ATP release increased 2- and 5-fold in the atria and 3 and 9-fold in the ventricles, respectively. In the presence of the N-type Ca2+ channel inhibitor -conotoxin (-CTX) (10 and 100 nM), the increase in the release of both NE and ATP in response to 50 mM K+ was abolished, indicating that this release was exocytotic.
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The P2XR antagonist PPADS (100 µM) and the ectonucleotidase inhibitor ARL67156(60 µM), alone or in combination, did not change basal NE release in atrial tissue (Fig. 3A). However, when porcine atrial tissue was depolarized with 50 mM K+, PPADS attenuated NE exocytosis as a function of its concentration (by 30 and 73%, at 30 and 100 µM, respectively; Fig. 3B). In contrast, in the presence of 60 µM ARL67156 NE exocytosis was enhanced by 27%, but again, PPADS attenuated it as a function of its concentration (by 50 and 80%, at 30 and 100 µM, respectively; Fig. 3B). PPADS (100 µM) did not modify basal or K+-stimulated (50 mM) ATP release (Fig. 3, C and D), whereas 60 µM ARL67156 either alone or in combination with 100 µM PPADS, doubled ATP release, both under control and K+-stimulated conditions (Fig. 3, C and D). Analogous to our observations in atrial tissue, neither the P2XR antagonist PPADS (100 µM) nor the E-NTPDase1/CD39 inhibitor ARL67156(60 µM) affected basal NE release in ventricular tissue (Fig. 4A). When porcine ventricular tissue was depolarized with 50 mM K+, PPADS attenuated NE exocytosis by 50 and 57%, at 30 and 100 µM, respectively (Fig. 4B). In the presence of ARL67156(60 µM), NE exocytosis was enhanced by 45%, but PPADS markedly reduced it (by 47 and 60%, at 30 and 100 µM, respectively; Fig. 4B). PPADS (100 µM) did not modify basal or K+-stimulated (50 mM) ATP release (Fig. 4, C and D), whereas 60 µM ARL67156 either alone or in combination with 100 µM PPADS, doubled ATP release, both in control and K+-stimulated conditions (Fig. 4, C and D). PPADS did not modify the increase in ATP release caused by ARL67156(Fig. 4, C and D).
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As shown in Fig. 5A, incubation with solCD39 markedly decreased both basal (90%, at 10 µM) and K+-stimulated (50 mM) ATP release (50 and 80%, at 1 and 10 µM, respectively) in porcine atrial tissue. In the same preparations, 10 µM solCD39 did not modify basal NE release but reduced K+-induced NE exocytosis by 45% (Fig. 5C). Analogous to atrial tissue, incubation with solCD39 markedly decreased both basal (73%, at 10 µM) and K+-stimulated (50 mM) ATP release (70 and 92%, at 1 and 10 µM, respectively) in ventricular tissue (Fig. 5B). In the same ventricular preparations, 10 µM solCD39 did not modify basal NE release, but it reduced the K+-induced NE exocytosis by 22% at 0.1 µM and by 45% at both 1 and 10 µM (Fig. 5D).
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Shown in Fig. 6 is the relationship between the release of ATP and NE in porcine heart tissue in response to K+ depolarization, either under control conditions or in the presence of PPADS, ARL67156 and solCD39. Thus, we have demonstrated a highly significant correlation between the K+-evoked release of ATP and that of NE, independently of whether ATP release was enhanced by 60 µM ARL67156or was diminished by incubation with 10 µM solCD39. In contrast, in the presence of 100 µM PPADS, NE release remained constant independently of the changes in ATP released. This lack of correlation in the presence of PPADS suggested that blockade of P2XR prevented ATP-induced modulation of NE release.
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Discussion |
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; Sneddon et al., 1999) and central sympathetic system (Poelchen et al., 2001) and also modulates release of other transmitters, such as NE (Burnstock, 1999). Using a synaptosomal preparation, i.e., a high-speed pellet of a collagenase-treated guinea pig heart homogenate containing "pinched-off" sympathetic nerve terminals, we proposed that, once released from these nerve endings, ATP amplifies NE release via a positive feedback mechanism initiated by P2XR activation (Sesti et al., 2002). We hypothesized that this ATP-dependent amplification of NE release could be influenced by E-NTPDase1/CD39, an enzyme that inactivates ATP, thereby interrupting the ATP-initiated positive feedback loop (Sesti et al., 2002). Whether E-NTPDase1/CD39 is localized to cardiac neurons and modulates NE exocytosis in intact cardiac tissue remained untested. The present investigation was designed to answer these questions.
We have now directly demonstrated the presence of E-NTPDase1/CD39 in cardiac nerves by immunohistochemistry. To confirm that E-NTPDase1/CD39 is localized in cardiac nerves, we used colocalization methodology using an anti-synapsin antibody to identify neuronal structures (De Camilli et al., 1983) and a mouse monoclonal antibody directed against human CD39. With this technique, we determined that in human and in porcine cardiac tissue, specific CD39 staining is selectively limited to neurons (Fig. 1). Notably, the staining could include both membrane-fixed (Gordon, 1986; Zimmermann and Braun, 1999) and releasable nucleotidases (Todorov et al., 1997; Westfall et al., 2002). Colocalization of CD39 with synaptophysin, demonstrating the presence of CD39 in neurons, had been previously reported in rat brain (Wang and Guidotti, 1998).
The presence of NTPDase1 and 2 was previously reported in murine cardiac tissues (Sevigny et al., 2002). NTPDase1 was localized in the endothelium, whereas NTPDase2 was found in pericytes (Sevigny et al., 2002). Also, mRNA for CD39L2 (NTPDase6) was found to be expressed in myocytes and capillary endothelial cells in human heart (Yeung et al., 2000). In the present study, using an anti-NTPDase1 antibody, we found immunostaining only in neurons in human and porcine cardiac tissue. The lack of staining in the microvasculature may be due to species differences. Although we did not search for other nucleotidases (e.g., NTPDase2), there are no reports in the literature of NTPDase2 in human and porcine heart.
Because of availability and similarity to human heart (Appel et al., 2001), we chose to use porcine cardiac tissue to investigate the role of neuronal E-NTPDase1/CD39 in cardiac adrenergic transmission. We determined that K+-evoked depolarization of porcine cardiac tissue, both atrial and ventricular, initiated release of both NE and ATP, and we verified that such release was inhibited by the N-type Ca2+ channel blocker -CTX (Sher et al., 1991), thereby demonstrating its exocytotic nature (Fig. 2).
The inter-relationship between the release of the two mediators (ATP and NE), and their modulation by E-NTPDase1/CD39, were assessed using 1) PPADS, a pharmacological agent that selectively blocks purinergic P2XR (Lambrecht, 2000); 2) ARL67156 a selective inhibitor of E-NTPDase1/CD39 (Crack et al., 1995); and 3) solCD39, which can exogenously supplement NTPDase activity (Sesti et al., 2002). We found that blockade of P2XR with PPADS did not affect ATP exocytosis in either atrial or ventricular tissue, thus excluding the possibility that ATP might modulate its own release via P2XR. In contrast, inhibition of E-NTPDase1/CD39 with ARL67156markedly potentiated the release of ATP, demonstrating that E-NTPDase1/CD39 is required for metabolic disposition of released ATP and thereby determines ATP availability at sympathetic nerve endings. Consistent with our results, ARL 67156 potentiates electrically evoked NE overflow in rat vas deferens, whereas PPADS inhibits it (Queiroz et al., 2003).
Notably, we found that the magnitude of NE exocytosis correlated positively with the quantities of ATP released. Indeed, when exocytosed ATP levels were increased by inhibition of E-NTPDase1/CD39 with ARL67156 NE exocytosis also increased; conversely, when the quantities of released ATP were diminished due to metabolism by exogenous solCD39, NE exocytosis was markedly reduced. The correlation between ATP and NE release thus remained constant during various experimental conditions, irrespective of whether ATP release was increased or decreased (Fig. 6). This correlation suggested that released ATP regulates NE exocytosis by activating presynaptic P2XR, as noted in previous studies (Boehm, 1999; Sperlágh et al., 2000; Sesti et al., 2002, 2003; Queiroz et al., 2003). Indeed, we found that in the presence of the P2XR antagonist PPADS, the curve relating NE release to that of ATP was shifted downwards, demonstrating that for the same quantities of ATP much less NE was released (Fig. 6). This strengthens the hypothesis that coreleased ATP modulates NE exocytosis via P2XR activation.
The important regulatory role played by E-NTPDase1/CD39 in P2XR-mediated modulation of NE exocytosis is clearly established by our results. Inhibition of this ectonucleotidase with ARL67156potentiated the release of both ATP and NE, whereas administration of solCD39 depressed it. Excessive NE release is a major cause of arrhythmic and coronary vascular dysfunction in myocardial ischemia (Braunwald and Sobel, 1988; Benedict et al., 1996; Levi and Smith, 2000). By curtailing NE release, in addition to its antithrombotic effects (Marcus et al., 2003), solCD39 may offer a novel therapeutic approach to ischemic clinical conditions. Indeed, our previous data indicated that solCD39 reduces ischemia-induced efflux of NE not only in synaptosomal preparations but also in the intact perfused heart (Sesti et al., 2003). This signifies that the circulation of the heart is in intimate contact with its neuronal system, raising the possibility that systemically administered high molecular weight agents affecting neuronal activity, such as solCD39, may induce immediate and beneficial effects under conditions of ischemia. As a therapeutic agent, solCD39 is likely to be more advantageous than a P2XR antagonist. Unlike P2XR antagonists, solCD39 will decrease ATP concentrations at sympathetic nerve endings, thus not only reducing activation of facilitatory P2X receptors but also favoring activation of low-threshold inhibitory P2Y receptors (Boehm and Kubista, 2002; Sesti et al., 2002).
Another possible application of solCD39 is in congestive heart failure. This disorder is usually accompanied by enhanced adrenergic discharge in the arteriolar bed. This results in an increase in peripheral resistance and afterload, producing further deterioration of cardiac function (Benedict et al., 1996; Esler and Kaye, 1998). By limiting excessive NE release, solCD39 should be a valuable adjunct in the management of cardiac failure.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: NE, norepinephrine; P2XR, purinergic P2X receptor(s); E-NTPDase1, ectonucleoside triphosphate diphosphohydrolase1; solCD39, recombinant, soluble form of human E-NTPDase1/CD39; KHS, Krebs-Henseleit solution; ARL67156 6-N,N-diethyl-
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-dibromomethylene-D-adenosine-5'-triphosphate; PPADS, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid; -CTX, -conotoxin; PBS, phosphate-buffered saline; Ab, antibody; ANOVA, analysis of variance.
Address correspondence to: Dr. Roberto Levi, Department of Pharmacology, 1300 York Ave., Room LC419, Weill Medical College of Cornell University, New York, NY 10021. E-mail rlevi{at}med.cornell.edu
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) represent basal release, triangles (
) are experiments performed in the presence of 50 mM K+, squares (
) are results from experiments performed in the presence of 50 mM K+ and ARL67156 and diamonds (
) represent data from experiments performed in the presence of 50 mM K+ and solCD39. Lines were calculated by linear regression analysis. The correlation coefficient was 0.889 (P < 0.001) for the solid line and 0.001 for the dotted line, showing that in the presence of PPADS, when P2XR are blocked, there is no correlation between NE and ATP release.