RAPID COMMUNICATION |
Correspondence to: M.R. Boyett, School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UK. E-mail: m.r.boyett@leeds.ac.uk
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adenosine exerts multiple receptor-mediated effects in the heart, including a negative chronotropic effect on the sinoatrial node. The aim of this study was to investigate the distribution of the equilibrative nucleoside transporter rENT1 in rat sinoatrial node and atrial muscle. Immunocytochemistry and/or immunoblotting revealed abundant expression of this protein in plasma membranes of sinoatrial node and in atrial and ventricular cells. Because rENT1-mediated transport is likely to regulate the local concentrations of adenosine in the sinoatrial node and other parts of the heart, it represents a potential pharmacological target that might be exploited to ameliorate ischemic damage during heart surgery. (J Histochem Cytochem 50:305309, 2002)
Key Words: sinoatrial node, adenosine, rENT1, nucleoside, transport
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
UPTAKE OF NUCLEOSIDES, such as adenosine, across the plasma membrane of mammalian cells is catalyzed both by equilibrative (Na+-independent) nucleoside transporters of the ENT family and concentrative (Na+-dependent) nucleoside transporters of the CNT family (
Nucleoside transporters play an important regulatory role in the adenosine-mediated regulation of many physiological processes, including coronary blood flow and myocardial O2 supplydemand balance. In the heart, adenosine regulates pacemaking and contractility through binding to cell surface purinergic receptors. In the pacemaker of the heart (the sinoatrial or SA node), adenosine has a negative chronotropic effect in a number of different species, including humans (
Previous studies have provided evidence for the existence of an es-type NBMPR-sensitive nucleoside transport process in isolated rat cardiac myocytes (e.g.,
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The antibodies used in this study were anti-rENT1226291 and anti-connexin (Cx43). The anti-rENT1 polyclonal antibody was raised in rabbit against a glutathione-S-transferase fusion protein bearing the central cytoplasmic loop region of rENT1 (residues 226291;
Immunocytochemical experiments were carried out on four rats, using tissue sections through the SA node and its surrounding atrial muscle. Rats of either sex weighing 0.20.3 kg were sacrified by stunning and cervical dislocation. Dissection and preparation for cryosectioning of the SA node and surrounding atrial muscle were carried out as previously described (
The specificity of the anti-rENT1 antibody was determined by immunoblotting of the intercaval (i.e., SA node-containing) region and atrial and ventricular tissue samples from seven rat hearts. Tissue samples were crushed under liquid N2 and then re-suspended in sample buffer (containing 1% SDS and 300 mM sucrose plus 10 mM EDTA, 0.1 mM iodoacetamide, 0.1 mM benzathonium chloride, and 0.57 mM phenylsulfonyl fluoride as protease inhibitors). After centrifugation at 10,000 x g for 34 min, samples (25 µg) of membrane myocardial proteins in the supernantant were separated by electrophoresis on 10% SDS polyacrylamide gels and then transferred to nitrocellulose membranes by semi-dry blotting. After blocking overnight at 4C in 10% dried skimmed milk powder, blots were incubated for 1 hr with 1.25 µg/ml anti-rENT1 antibody in PBS containing 0.05% Tween-20. The bound antibody was detected with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:30,000; Dako, Poole, UK). Immunoreactivity was visualized by using a peroxidase-based chemiluminescent substrate kit (Amersham Life Sciences; Poole, UK).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To assess the distribution of rENT1 in the heart, sections of the rat SA node and its surrounding atrial muscle were simultaneously probed with rabbit anti-rENT1 and mouse Cx43 antibodies. The anti-Cx43 antibody was used as a negative morphological marker for the SA node, as previously described (
|
Immunostaining revealed that rENT1 was also abundant in the atrial muscle of the crista terminalis adjoining the SA node (Fig 1C). As in the case of SA node cells, immunostaining was located predominantly at the plasma membrane, and rENT1 labeling intensity within the crista terminalis (Fig 1C) was comparable with that seen in the SA node (Fig 1A). The atrial cells of the crista terminalis stained strongly for Cx43 (Fig 1D), as expected from previous studies (e.g.,
The specificity of the rENT1 staining seen in sections of the SA node and crista terminalis was evident from the lack of staining obtained if the primary antibody was omitted or if it was pre-absorbed with the CBD fusion protein bearing the central cytoplasmic region (residues 226291) of rENT1 (data not shown). Additional evidence for the specificity of the anti-rENT1 antibody was provided by immunoblotting of membrane preparations from rat heart; tissue was taken from the intercaval region, where the SA node is located, atrium, and ventricle. In each case the antibody stained primarily a single band of apparent molecular mass approximately 60 kD (Fig 2). Similar results were obtained from seven rats. Although the size of rENT1, predicted from its amino acid sequence, is 50 kD, photoaffinity labeling studies have revealed that the es-type transporter from various rat tissues migrates as an N-glycosylated species with apparent molecular mass of 62 kD (
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cardiac Automaticity
Because nucleoside transporters exert effects on the local concentration of adenosine in tissues, the presence of the es-type transporter rENT1 in the rat SA node is likely to be of physiological importance. Pharmacological evidence for this importance has been provided in guinea pig SA and AV nodes, where dipyridamole, a potent inhibitor of es-type transporters in this species, was found to potentiate the chronotropic and dromotropic effects of adenosine, respectively (
Preconditioning and Ischemia
Adenosine has been suggested to have a cardioprotective role in myocardial ischemia. Myocardial ischemia can be defined as "an imbalance between the amount of oxygen and substrates supplied to the heart and the amount needed to perform normal function" (
![]() |
Acknowledgments |
---|
Supported by the British Heart Foundation, the MRC, the Wellcome Trust, the Canadian Institutes of Health Research, and the Alberta Cancer Board. Fatima Abidi was supported by an ORS award. Carol Cass is Canada Research Chair in Oncology.
Received for publication October 25, 2001; accepted November 21, 2001.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baldwin SA, Mackey JR, Cass CE, Young JD (1999) Nucleoside transporters: molecular biology and implications for therapeutic development. Mol Med Today 5:216-224[Medline]
Belardinelli L, Linden J, Berne RM (1989) The cardiac effects of adenosine. Prog Cardiovasc Dis 32:73-97[Medline]
de Jong JW, de Jonge R, Keijzer E, Bradamate S (2000) The role of adenosine in preconditioning. Pharmacol Ther 87:141-149[Medline]
Dobrzynski H, Rothery SM, Marples DR, Coppen SR, Takagishi Y, Honjo H, Tamkun MM, Henderson Z, Kodama I, Severs NJ, Boyett MR (2000) Presence of the Kv1.5 K+ channel in the sinoatrial node. J Histochem Cytochem 48:769-780
Geisbuhler TP, Johnson DA, Rovetto MJ (1987) Cardiac myocyte guanosine transport and metabolism. Am J Physiol 253:C645-651
Griffith DA, Jarvis SM (1996) Nucleoside and nucleobase transport systems of mammalian cells. Biochim Biophys Acta 1286:153-181[Medline]
Griffiths M, Beaumont N, Yao SYM, Sundaram M, Boumah CE, Davies A, Kwong FYP, Coe I, Cass CE, Young JD, Baldwin SA (1997) Cloning of a human nucleoside transporter implicated in the cellular uptake of adenosine and chemotherapeutic drugs. Nature Med 3:89-93[Medline]
Hakes DJ, Dixon JE (1992) New vectors for high level expression of recombinant proteins in bacteria. Anal Biochem 202:293-298[Medline]
Lee HT, LaFaro RJ, Reed GE (1995) Pre-treatment of human myocardium with adenosine during open heart surgery. J Cardiovasc Surg 10:665-676
Meester BJ, Shankley NP, Welsh NJ, Meijler FL, Black JW (1998) Pharmacological analysis of the activity of the adenosine uptake inhibitor, dipyridamole, on the sinoatrial and atrioventricular nodes of the guinea-pig. Br J Pharmacol 124:729-741[Abstract]
Murry CE, Jennings RB, Reimer KA (1986) Preconditioning with ischaemia: a delay of lethal cell injury in ischaemic myocardium. Circulation 74:1124-1136[Abstract]
Rongen GA, Smits P, Donck KV, Willemsen JJ, Deabreu RA, Van Belle H, Thien T (1995) Hemodynamic and neurohumoral effects of various grades of selective adenosine transport inhibition in humansimplications for its future role in cardioprotection. J Clin Invest 95:658-668[Medline]
Sundaram M, Yao SYM, Ng AML, Griffiths M, Cass CE, Baldwin SA, Young JD (1998) Chimeric constructs between human and rat equilibrative nucleoside transporters (hENT1 and rENT1) reveal hENT1 structural domains interacting with coronary vasoactive drugs. J Biol Chem 273:21519-21525
Verdouw PD, van den Doel MA, de Zeeuw S, Duncker DJ (1998) Animal models in the study of myocardial ischaemia and ischaemic syndromes. Cardiovasc Res 39:121-135[Medline]
Ward JL, Sherali A, Mo Z-P, Tse C-M (2000) Kinetic and pharmacological properties of cloned human equilibrative nucleoside transporters, ENT1 and ENT2, stably expressed in nucleoside transporter-deficient PK15 cells. ENT2 exhibits a low affinity for guanosine and cytidine but a high affinity for inosine. J Biol Chem 275:8375-8381
West GA, Belardinelli L (1985) Sinus slowing and pacemaker shift caused by adenosine in the rabbit SA node. Pflugers Arch 403:66-74[Medline]
Yao SY, Ng AM, Muzyka WR, Griffiths M, Cass CE, Baldwin SA Young JD (1997) Molecular cloning and functional characterization of nitrobenzylthioinosine (NBMPR)-sensitive (es) and NBMPR-insensitive (ei) equilibrative transporter proteins (rENT1 and rENT2) from rat tissue. J Biol Chem 272:28423-28430
Zaza A, Rocchetti M, DiFrancesco D (1994) Modulation of the hyperpolarization activated current (If) by adenosine in the rabbit sino-atrial node. Circulation 94:734-741