formycin and 4-nitrobenzylthioinosine

1. Microvascular endothelial cells (MVECs) form a barrier between circulating metabolites, such as adenosine, and the surrounding tissue. We hypothesize that MVECs have a high capacity for the accumulation of nucleosides, such that inhibition of the endothelial nucleoside transporters (NT) would profoundly affect the actions of adenosine in the microvasculature. 2. We assessed the binding of [(3)H]nitrobenzylmercaptopurine riboside (NBMPR), a specific probe for the inhibitor-sensitive subtype of equilibrative NT (es), and the uptake of [(3)H]formycin B (FB), by MVECs isolated from rat skeletal muscle. The cellular expression of equilibrative (ENT1, ENT2, ENT3) and concentrative (CNT1, CNT2, CNT3) NT subtypes was also determined using both qualitative and quantitative polymerase chain reaction techniques. 3. In the absence of Na(+), MVECs accumulated [(3)H]FB with a V(max) of 21+/-1 pmol microl(-1) s(-1). This uptake was mediated equally by es (K(m) 260+/-70 microm) and ei (equilibrative inhibitor-insensitive; K(m) 130+/-20 microm) NTs. 4. A minor component of Na(+)-dependent cif (concentrative inhibitor-insensitive FB transporter)/CNT2-mediated [(3)H]FB uptake (V(i) 0.008+/-0.005 pmol microl(-1) s(-1) at 10 microm) was also observed at room temperature upon inhibition of ENTs with dipyridamole (2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido-[5,4-d]pyrimidine)/NBMPR. 5. MVECs had 122,000 high-affinity (K(d) 0.10 nm) [(3)H]NBMPR binding sites (representing es transporters) per cell. A lower-affinity [(3)H]NBMPR binding component (K(d) 4.8 nm) was also observed that may be related to intracellular es-like proteins. 6. Rat skeletal muscle MVECs express es/ENT1, ei/ENT2, and cif/CNT2 transporters with characteristics typical of rat tissues. This primary cell culture model will enable future studies on factors influencing NT subtype expression, and the consequent effect on adenosine bioactivity, in the microvasculature.

Topics: Animals; Capillaries; Cell Separation; Cells, Cultured; Dilazep; Dipyridamole; DNA Primers; Endothelial Cells; Formycins; Muscle, Skeletal; Nucleoside Transport Proteins; Piperazines; Radioligand Assay; Rats; Reverse Transcriptase Polymerase Chain Reaction; Thioinosine; Vasodilator Agents

Adenosine uptake via nucleoside transporters is inhibited when S49 and NG108-15 cell lines cells are exposed to ethanol. This inhibition leads to an accumulation of extracellular adenosine that binds to adenosine A2 receptors and increases cAMP production. Subsequently, there is a heterologous desensitization of receptors coupled to adenylyl cyclase for which adenosine also is required. There are multiple classes of facilitative and concentrative nucleoside transporters that could be inhibited by ethanol to initiate this cascade of events. In this paper, we establish that adenosine uptake by only one type of nucleoside transporter, an NBMPR-sensitive facilitative transporter, is inhibited by ethanol. There is no effect on other classes of nucleoside transporters even when present in the same cell. Thus, ethanol-induced extracellular accumulation of adenosine results specifically from inhibition of NBMPR-sensitive facilitative nucleoside transporters. We also find that human lymphocytes express only facilitative nucleoside transporters and that the NBMPR-sensitive type is predominant. Thus, inhibition of this type of transporter by ethanol may be related to the desensitization of cAMP signal transduction that we have reported in lymphocytes from alcoholics.

Topics: Adenosine; Adult; Biological Transport; Blood Proteins; Carrier Proteins; Ethanol; Formycins; Glutamates; Glutamic Acid; Humans; In Vitro Techniques; Isoleucine; Lymphocytes; Male; Membrane Proteins; Middle Aged; Nucleoside Transport Proteins; Sodium; Thioinosine

A single dose of the adenosine analogue Formycin A (FoA) (20 mg/kg), combined with nitrobenzyl mercaptopurine ribonucleoside 5'-monophosphate (NBMPR-P) (10 mg/kg), a prodrug of nitrobenzylthioinosine (NBMPR), was effective in reducing the size of the foot pad lesions from 7.4 +/- 0.2 to 3.9 +/- 0.2 of Syrian golden hamsters infected with Leishmania major. There was a statistical difference (p < 0.01) in the size of the foot pad by the fifth day between the infected groups that received treatment and the controls, as well as between the groups that were treated with combined drugs and FoA only. The initial reduction in size of the foot pad noted in the group that received only FoA was transient. The effect of FoA or FoA combined with NBMPR on the in vitro cultured promastigotes was similar, indicating that the transport inhibitor might be manipulating the availability of FoA in the host's macrophages where the leishmania amastigotes are resident. The results further indicate the need to explore the usefulness of combining cytotoxic nucleoside analogues with host protecting nucleoside transport inhibitors in the treatment of protozoan parasitic infections.

Topics: Animals; Cricetinae; Drug Combinations; Female; Foot; Formycins; Leishmania; Leishmaniasis; Mesocricetus; Microbial Sensitivity Tests; Thioinosine