L‐[³H]Adenosine, a New Metabolically Stable Enantiomeric Probe for Adenosine Transport Systems in Rat Brain Synaptoneurosomes

Abstract

The stereoenantimers D-[3H]adenosine and L-[3H]adenosine were used to study adenosine accumulation in rat cerebral cortical synaptoneurosomes. L-Adenosine very weakly inhibited rat brain adenosine deaminase (ADA) activity with a Ki value of 385 microM. It did not inhibit rat brain adenosine kinase (AK) activity, nor was it utilized as a substrate for either ADA or AK. The rate constants (fmol/mg of protein/s) for L-[3H]adenosine accumulation measured in assays where transport was stopped either with inhibitor-stop centrifugation or with rapid filtration methods were 82 +/- 14 and 75 +/- 10, respectively. Using the filtration method, the rates of L-[3H]adenosine accumulation were not significantly different from the value of 105 +/- 15 fmol/mg of protein/s measured for D-[3H]adenosine transport. Unlabeled D-adenosine and nitrobenzylthiolnosine, both at a concentration of 100 microM, reduced the levels and rates of L-[3H]adenosine accumulation by greater than 44%. These findings suggest that L-adenosine, a metabolically stable enantiomeric analog, and the naturally occurring D-adenosine are both taken up by rat brain synaptoneurosomes by similar processes, and as such L-adenosine may represent an important new probe with which adenosine uptake may be studied.

Kinetic and allosteric cooperativity in L-adenosine transport in chromaffin cells. A mnemonical transporter

“…The metabolic problem can also be solved by using nonmetabolizable analogs such as L-adenosine. The L-adenosine transport has been characterized in other cellular models, such as erythrocytes and synaptoneurosomal preparations (Gati et al, 1989;Gu et al, 1991, Gu & Geiger, 1992). In the present experimental work, we have characterized the transport of L-adenosine in a homogeneous neural cell population, the chromaffin cell.…”

The Enantioselectivity of Enzymes Involved in Current Antiviral Therapy Using Nucleoside Analogues: A New Strategy?

“…The enantioselectivity of nonenzymatic phenomena must also be considered. For example, nucleoside transport is an important parameter in the pharmacology of drug action, which exhibits enantiomeric selectivity as shown in the case of adenosine (Gati et al, 1989;Gu et al, 1991) and carbovir (Mahony et al, 1992). In spite of these adverse arguments and the fact that much work remains to be done, the indicative trends in enzyme enantioselectivity reported in this review may still be used as a coherent guide line to help in the search for new interesting L-nucleoside analogues.…”

“…Inhibition of ADA also proceeds with high enantioselectivity, since the (2S,3R) enantiomer of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) is a 250-fold more potent inhibitor of human erythrocyte ADA than the opposite enantiomer (Bessodes et al, 1982). β-L-Adenosine is only a weak inhibitor of rat brain ADA when the natural enantiomer is used as substrate (Gu et al, 1991). The high enantioselectivity of deamination cata-lysed by ADA finds its use in the resolution of racemic adenosine analogues in the presence of ADA: the enantiomer with the natural or pseudonatural configuration is always rapidly deaminated whereas the antipode is resistant.…”

“…β-L-2′,3′-Dideoxyadenosine and β-L-2′,3′-didehydro-2′,3′-dideoxyadenosine are very poor substrates and inhibitors of calf intestinal ADA contrary to the natural enantiomers (Pelicano et al, 1997). Several sources have reported that β-L-adenosine is not a substrate of ADA (Gu et al, 1991;Phadtare & Zemlicka, 1989) and that β-L-2′-deoxyadenosine is a very poor substrate compared to the D-enantiomer (Gaubert, 1999b). Inhibition of ADA also proceeds with high enantioselectivity, since the (2S,3R) enantiomer of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) is a 250-fold more potent inhibitor of human erythrocyte ADA than the opposite enantiomer (Bessodes et al, 1982).…”

“…β-L-Adenosine has been reported to be neither a substrate nor an inhibitor of rat brain AK (Gu et al, 1991). Bovine liver AK catalyses the phosphorylation of β-L-adenosine with a lower efficiency than that of its natural enantiomer and it displays only a very weak affinity for β-L-dA (Gaubert, 1999b).…”