The adenosine transporter,ENT1, in cardiomyocytes is sensitive to inhibition by ethanol in a kinase-dependent manner: implications for ethanol-dependent cardioprotection and nucleoside analog drug cytotoxicity

The adenosine transporter 1 (ENT1) transports nucleosides, such as adenosine, and cytotoxic nucleoside analog drugs. ENT1 is well established to play a role in adenosinergic signaling in the cardiovascular system by modulating adenosine levels. Moderate ethanol consumption is cardioprotective and underlying mechanisms of action are not clear although adenosinergic signaling has been implicated. Here, we show that ethanol (5–200 mM) significantly reduces ENT1-dependent [³H] 2-chloroadenosine uptake (by up to 27 %) in the cardiomyocyte cell line, HL-1. Inhibition or absence of ENT1 is known to be cardioprotective, suggesting that the interaction of ethanol with ENT1 may promote adenosinergic cardioprotective pathways in the cardiovasculature. Ethanol sensitivity of adenosine uptake is altered by pharmacological activation of PKA and PKC. Primary cardiomyocytes from PKCε-null mice have significantly greater sensitivity to inhibition (by approximately 37 %) of adenosine uptake by ethanol than controls. These data suggest that the presence of ethanol may compromise ENT1-dependent nucleoside analog drug cytotoxicity, and indeed, ethanol (5 mM) reduces the cytotoxic effects of gemcitabine (2 nM), an anti-cancer drug, in the human cancer cell line, HTB2. Thus, the pharmacological inhibition of ENT1 by ethanol may contribute to ethanol-dependent cardioprotection but compromise gemcitabine cytotoxicity.     

Apo and holo crystal structures of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans.

The aldehyde dehydrogenases (ALDHs) are a superfamily of multimeric enzymes which catalyse the oxidation of a broad range of aldehydes into their corresponding carboxylic acids with the reduction of their cofactor, NAD or NADP, into NADH or NADPH. At present, the only known structures concern NAD-dependent ALDHs. Three structures are available in the Protein Data Bank: two are tetrameric and the other is a dimer. We solved by molecular replacement the first structure of an NADP-dependent ALDH isolated from Streptococcus mutans, in its apo form and holo form in complex with NADP, at 1.8 and 2.6 A resolution, respectively. Although the protein sequence shares only approximately 30 % identity with the other solved tetrameric ALDHs, the structures are very similar. However, a large local conformational change in the region surrounding the 2' phosphate group of the adenosine moiety is observed when the enzyme binds NADP, in contrast to the NAD-dependent ALDHs.Structure and sequence analyses reveal several properties. A small number of residues seem to determine the oligomeric state. Likewise, the nature (charge and volume) of the residue at position 180 (Thr in ALDH from S. mutans) determines the cofactor specificity in comparison with the structures of NAD-dependent ALDHs. The presence of a hydrogen bond network around the cofactor not only allows it to bind to the enzyme but also directs the side-chains in a correct orientation for the catalytic reaction to take place. Moreover, a specific part of this network appears to be important in substrate binding. Since the enzyme oxidises the same substrate, glyceraldehyde-3-phosphate (G3P), as NAD-dependent phosphorylating glyceraldehyde-3-phosphate dehydrogenases (GAPDH), the active site of GAPDH was compared with that of the S. mutans ALDH. It was found that Arg103, Arg283 and Asp440 might be key residues for substrate binding.