Distribution and Functional Characterization of Equilibrative Nucleoside Transporter-4, a Novel Cardiac Adenosine Transporter Activated at Acidic pH

Abstract

Abstract-Adenosine plays multiple roles in the efficient functioning of the heart by regulating coronary blood flow, cardiac pacemaking, and contractility. Previous studies have implicated the equilibrative nucleoside transporter family member equilibrative nucleoside transporter-1 (ENT1) in the regulation of cardiac adenosine levels. We report here that a second member of this family, ENT4, is also abundant in the heart, in particular in the plasma membranes of ventricular myocytes and vascular endothelial cells but, unlike ENT1, is virtually absent from the sinoatrial and atrioventricular nodes. Originally described as a monoamine/organic cation transporter, we found that both human and mouse ENT4 exhibited a novel, pH-dependent adenosine transport activity optimal at acidic pH (apparent K m values 0.78 and 0.13 mmol/L, respectively, at pH 5.5) and absent at pH 7.4. In contrast, serotonin transport by ENT4 was relatively insensitive to pH. ENT4-mediated nucleoside transport was adenosine selective, sodium independent and only weakly inhibited by the classical inhibitors of equilibrative nucleoside transport, dipyridamole, dilazep, and nitrobenzylthioinosine. We hypothesize that ENT4, in addition to playing roles in cardiac serotonin transport, contributes to the regulation of extracellular adenosine concentrations, in particular under the acidotic conditions associated with ischemia. Key Words: nucleoside Ⅲ adenosine Ⅲ transport Ⅲ ischemia Ⅲ pH T he purine nucleoside adenosine is produced by the action of both endo-and ecto-nucleotidases on adenine nucleotides in the heart and plays key roles in the regulation of coronary blood flow and myocardial O 2 supply-demand balance. 1-4 For example, action of adenosine on A 2A receptors on vascular smooth muscle and endothelial cells causes coronary vasodilatation. 1,5 In contrast, the negative inotropic and dromotropic effects of adenosine on the heart are mediated primarily by A 1 receptors. 2 Similarly, the negative chromotropic effect of adenosine involves action of A 1 receptors in the sinoatrial (SA) node on the inwardly rectifying potassium channel current I K-Ado and the hyperpolarization-activated pacemaker current I f . 2,6 Endogenous adenosine, acting on mitochondrial K ATP channels via A 1 and A 3 receptors, also makes a major contribution to the phenomenon of ischemic preconditioning. 5,7 Extracellular adenosine concentrations in the heart are governed both by action of ecto-5Ј-nucleotidase on adenine nucleotides released from cells and by transporter-mediated flux of adenosine across cell membranes. 3,4 Although most adenosine production occurs intracellularly, under normoxic conditions, metabolism maintains a low intracellular concentration and, therefore, the net flux of adenosine is into cardiomyocytes and endothelial cells. Under such conditions, administration of transport inhibitors increases extracellular concentrations of adenosine, leading to vasodilatation. 8 However, increased adenine nucleotide breakdown and inhibition of adenosine kinase duri...

Anatomical Distribution of Nucleoside System in the Human Brain and Implications for Therapy

“…NBTI partially inhibits ENTs at the nM concentration range ("es": equilibrative, NBTI sensitive type of ENTs, e.g., ENT1), whereas NBTI insensitive transporters are inhibited by NBTI only at the m M concentration range ("ei": equilibrative, NBTI insensitive type of ENTs, e.g., ENT2). The concentrative nucleoside transporter family (CNT transporters; unidirectional, sodium-dependent) includes six CNT transporter types (N1-N6) that are classi fi ed based on the types of nucleosides transported and sodium transport coupling Barnes et al 2006 ;Jennings et al 2001 ;Parkinson et al 2011 ;Pennycooke et al 2001 ;Ritzel et al 2001 ) .…”

“…The levels of nucleosides in brain and spinal cord areas were compared to the grand average concentration values of the total brain and spinal cord areas (Kovács et al 2010a ) . We also listed some brain and spinal cord areas, which are implicated in particular CNS diseases, even though their nucleoside levels did not differ from average values (these brain areas are labeled by "+") References : 1 Kovács et al 2010a ;2 Nagata et al 1984 ;3 Norstrand et al 1984 ;4 Phillips and Newsholme 1979 ;5 Norstrand and Glantz 1980 ;6 Dawson 1971 ;7 Jennings et al 2001 ;8 Baldwin et al 2005 ;9 Barnes et al 2006 ;10 Pennycooke et al 2001 ;11 Ritzel et al 2001 ;12 Svenningsson et al 1997 ;13 Fredholm et al 2001 ; Abbreviations : Nucleosides-Ado Adenosine, Guo Guanosine, Ino Inosine, Urd Uridine; Nucleoside metabolizing enzymes-5 ¢ NT 5 ¢ -Nucleotidase, ADA Adenosine deaminase, ADK Adenosine kinase, GDA Guanine deaminase, PNP Purine nucleoside phosphorylase. For nucleoside transporter and nucleoside receptor abbreviations, see text …”

“…The distribution of nucleoside transporters in the human brain is uneven, and the regionally different distribution of nucleoside transporters re fl ects the functional signi fi cance of nucleoside neuromodulation in different brain areas Barnes et al 2006 ;Jennings et al 2001 ;Pennycooke et al 2001 ;Ritzel et al 2001 ) .…”

“…Nucleoside transporters are present in the caudate nucleus, putamen, globus pallidus, and substantia nigra (Barnes et al 2006 ;Jennings et al 2001 ;Ritzel et al 2001 ) . Caudate nucleus, putamen, and globus pallidus contain high levels of A 1 and A 2A Rs (Jennings et al 2001 ) .…”

“…Nucleoside levels, metabolic enzymes, transporters, and receptors are unevenly distributed in the human brain ; Barnes et al 2006 ;Dawson 1971 ;Fredholm et al 2001 ;Jennings et al 2001 ;Kovács et al 1998Kovács et al , 2010aNagata et al 1984 ;Norstrand et al 1984 ;Norstrand and Glantz 1980 ;Pennycooke et al 2001 ;Phillips and Newsholme 1979 ;Ritzel et al 2001 ) . In addition, nucleoside concentrations are dependent on age and gender (Kovács et al 2010b ) .…”