Sodium-dependent and equilibrative nucleoside transport systems in L1210 mouse leukemia cells: effect of inhibitors of equilibrative systems on the content and retention of nucleosides.

The presence of 10 microM dipyridamole in incubation media of L1210/C2 cells decreased initial rates of zero-trans influx of formycin B (FB, 50 microM), a poorly metabolized inosine analogue, from 4.84 pmol/microliters cell water/s to 0.87 pmol/microliter cell water/s. However, after a 5-min interval of uptake, free FB levels in dipyridamole-treated cells were 165 pmol/microliters cell water, 2.3-fold greater than in dipyridamole-free cultures. This indicated the presence of a concentrative, dipyridamole-insensitive nucleoside transport (NT) system in L1210 cells, in addition to the equilibrative NT systems known to be expressed in these cells. The concentrative system was demonstrable only in the presence of NT inhibitors and required extracellular Na+. The presence of 8 microM 6-[(4-nitrobenzyl)thio]-9-beta-D- ribofuranosylpurine or 15 microM dilazep also induced an accumulation of free FB above steady-state levels, although of a lesser magnitude than that observed with dipyridamole. It appears that NT inhibitors induced nucleoside accumulation by inhibiting bidirectional nucleoside movements mediated by the equilibrative component of nucleoside transport in L1210/C2 cells without interfering with inward FB fluxes mediated by the Na(+)-dependent transporter. The presence of NT inhibitors also enhanced the cellular accumulation and retention of arabinosyladenine and its 5'-triphosphate in these cells. The increased cellular accumulation of 9-beta-D-arabinofuranosyladenine and 9-beta-D-arabinofuranosyladenine triphosphate by dipyridamole was associated with enhanced antiproliferative activity of 9-beta-D-arabinofuranosyladenine towards the leukemia cells.

Characterization of Na(+)-dependent, active nucleoside transport in rat and mouse peritoneal macrophages, a mouse macrophage cell line and normal rat kidney cells.

Peritoneal rat macrophages expressed solely an Na(+)-dependent, concentrative nucleoside transporter, which possesses a single Na(+)-binding site and transports purine nucleosides and uridine but not thymidine or deoxycytidine. The Michaelis-Menten constants for formycin B and Na+ were about 6 microns and 14 mM, respectively, and the estimated Na+:formycin B stoichiometry was 1:1. Rat macrophages accumulated 5 microM formycin B to a steady-state level exceeding that in the medium by about 500-fold during 60 min of incubation at 37 degrees C. Concentrative formycin B transport was resistant to inhibition by nitrobenzylthioinosine, lidoflazine, dilazep and nifedipine, but was slightly inhibited by high concentrations of dipyridamole (greater than 10 microM) and probenecid (greater than 100 microM). Mouse peritoneal macrophages and lines of mouse macrophages and normal rat kidney cells expressed Na(+)-dependent, active nucleoside transport but in addition significant Na(+)-independent, facilitated nucleoside transport. Facilitated nucleoside transport in these cells was sensitive to inhibition by nitrobenzylthioinosine, dilazep and dipyridamole. The presence of these inhibitors greatly enhanced the concentrative accumulation of formycin B by these cells by inhibiting the efflux via the facilitated transporter of the formycin B actively transported into the cells. Whereas rat macrophages lacked high-affinity nitrobenzylthioinosine-binding sites, mouse macrophages and normal rat kidney cells possessed about 10,000 such sites/cell. Rat and mouse erythrocytes, rat lymphocytes, and lines of Novikoff rat hepatoma cells, Chinese hamster ovary cells, Mus dunni cells and embryonic monkey kidney cells expressed only facilitated nucleoside transport.

Na(+)-dependent, active and Na(+)-independent, facilitated transport of formycin B in mouse spleen lymphocytes.

Na(+)-dependent, active and Na(+)-independent facilitated nucleoside transport were characterized in mouse spleen cells using rapid kinetic techniques and formycin B, a metabolically inert analog of inosine, as substrate. The Michaelis-Menten constants for formycin B transport by the two transporters were about 30 and 400 microM, respectively. The first-order rate constant for Na(+)-dependent transport was about 4-times higher than that for facilitated formycin B transport. The Na(+)-dependent carrier is specific for uridine and purine nucleosides and accumulates formycin B concentratively in an unmodified form. Concentrative accumulation was inhibited by ATP depletion and gramicidin and ouabain treatment of the cells. Our data indicate a single Na(+)-binding site on the Na(+)-dependent nucleoside carrier and a Michaelis-Menten constant for Na+ of about 10 mM. This transporter was not significantly inhibited by dipyridamole and nitrobenzylthioinosine, inhibitors of the facilitated transporter. The Na(+)-independent, facilitated nucleoside transporter of spleen cells exhibits properties comparable to those of the carriers present in mammalian cells in general. The B lymphocytes remaining after depletion of spleen cell populations of T lymphocytes by incubation with a combination of T-cell specific monoclonal antibodies plus complement exhibited about the same activities of active and facilitated nucleoside transport as the original suspension.

Cloning, genomic organization and chromosomal localization of the gene encoding the murine sodium-dependent, purine-selective, concentrative nucleoside transporter (CNT2).

A PCR-based strategy was used to isolate a 2653 bp cDNA encoding the mouse sodium-dependent, purine nucleoside selective, concentrative nucleoside transporter (designated mCNT2). The deduced protein sequence exhibits 93 and 80% identity to the previously cloned rat and human sodium-dependent, purine nucleoside selective, nucleoside transporters, respectively. Characterization of 3H-nucleoside uptake by COS-1 cells transiently transfected with the cDNA demonstrated that it encoded a functional nucleoside transport activity with selectivity for purine nucleosides. The cDNA was used to screen a murine (strain 129SvJ/6) genomic library in pBeloBAC11 to identify a clone containing the mCNT2 gene. A PCR strategy was used to identify and sequence the intron-exon boundaries and to determine the approximate sizes of the introns. The mCNT2 gene spans approximately 13.7 kb and is encoded by 15 exons. The gene was mapped to mouse chromosome 2e3 by fluorescence in situ hybridization.

Effect of cellular differentiation on nucleoside transport in human neuroblastoma cells.

The nucleoside transport characteristics of undifferentiated and differentiated LA-N-2 human neuroblastoma cells were compared through measurement of the cellular accumulation of [3H]formycin B in the absence and presence of specific nucleoside transport blockers such as dipyridamole and nitrobenzylthioinosine (NBMPR). [3H]NBMPR was also used as a high affinity probe to obtain an estimate of the number of NBMPR-sensitive nucleoside transport proteins. Undifferentiated LA-N-2 cells accumulated [3H]formycin B (25 microM) via a NBMPR/dipyridamole sensitive, Na(+)-independent, nucleoside transport system (Vi = 1.52 pmol/microliters/s; maximum intracellular concentration = 45 pmol/microliters cell water). The undifferentiated cells also had a high density of site-specific [3H]NBMPR binding sites (135,000 sites/cell; KD = 0.4 nM). When cell differentiation was induced by exposure to a serum-free defined medium, the initial rate of transporter-mediated [3H]formycin B uptake increased to 1.92 pmol/microliters/s, and the steady-state intracellular concentration of [3H]formycin B also increased significantly to 73 pmol/microliters. However, there was no concomitant change in the number of [3H]NBMPR binding sites, and the additional uptake was not Na(+)-dependent. This enhanced uptake in the differentiated cells appeared to be due, in part, to an increased functional expression of a NBMPR-resistant form of facilitated nucleoside transporter. Approximately 18% of the transporter-mediated uptake in the differentiated cells was resistant to inhibition by NBMPR at concentrations that blocked transport completely in the undifferentiated cells. This cell model may prove useful for basic studies on regulation of nucleoside transporter subtype expression in neural tissues, and for evaluation of the efficacy and potential host toxicity of cytotoxic nucleoside analogues (+/- specific transport blockers) in the treatment of neuroblastoma.

Temperature dependence of the release of ATP hydrolysis products from the 10S conformation of smooth muscle myosin.

The transition of smooth muscle myosin to the folded 10S monomeric conformation dramatically inhibits the release of the ATP hydrolysis products, ADP and Pi. In this work, we examined the influence of temperature on the time course of product release from the 10S conformer of chicken gizzard smooth muscle myosin. Release was monitored by single turnover assays, using either [gamma-32P]ATP or the fluorescent ATP analog, formycin triphosphate (FTP). For all temperatures over the range 15-35 degrees C, single exponential kinetics described the observed product release from 10S myosin. A 10 degrees C increase in temperature resulted in a fourfold increase in the rate constant for the observed product release. Using single turnover analysis, we found a similar temperature dependence for the apparent rate constants for product release from the extended 6S monomeric conformation of myosin. However, at any given temperature, the rate constant for 6S myosin was approximately 1.5 orders of magnitude greater than that for the 10S. These results are consistent with a kinetic scheme in which 10S myosin must undergo transition to the 6S conformation prior to product release.

Equilibrative adenosine transport in rat hepatocytes after chronic ethanol feeding.

Acute treatment of cells with ethanol in vitro inhibits adenosine uptake via equilibrative nucleoside transporters. After longer periods of exposure to ethanol in culture, rechallenge with ethanol no longer inhibits adenosine uptake. Herein, we have investigated the long-term effects of ethanol consumption in vivo on equilibrative nucleoside transport. Rats were fed a liquid diet containing 35% of calories as ethanol (ethanol-fed). Control rats were pair-fed a liquid diet that isocalorically substituted maltose dextrins for ethanol. After 4 weeks of ethanol consumption, nucleoside transport was measured in isolated hepatocytes. Uptake of [3H]adenosine was lower in ethanol-fed rats compared with control. Influx of the nonmetabolizable nucleoside analog, [3H]formycin B, was also decreased after ethanol feeding. However, neither the number of nitrobenzylthioinosine (NBMPR) binding sites or inhibition of adenosine uptake by NBMPR were affected by ethanol feeding. In controls, acute treatment of isolated hepatocytes with 100 mM ethanol inhibited [3H]adenosine uptake by 30-40%. However, in ethanol-fed rats, acute challenge with ethanol did not inhibit [3H]adenosine uptake. These data demonstrate that long-term ethanol feeding decreases equilibrative nucleoside transport in hepatocytes independent of a change in the number of nucleoside transporters and renders adenosine uptake insensitive to inhibition by ethanol.

Sodium-dependent nucleoside transport in rabbit intestinal epithelium.

Cellular uptake of formycin B, a poorly metabolized analog of inosine, by the isolated epithelium of rabbit jejunum is three times higher in the presence of Na+ than without this cation. The Na(+)-dependent nucleoside transport system is located in the apical membrane of the enterocytes and is capable of uphill transport, as shown for formycin B and adenosine with brush border membrane vesicles. According to present and earlier evidence, nucleoside transport across the basolateral membrane appears to have the properties of facilitated diffusion. Na(+)-dependent formycin B transport activity in intestinal epithelium decreases from jejunum to ileum and is absent in descending colon. As with Na(+)-coupled cotransport systems for other organic solutes, apical entry of formycin B is driven by the electrochemical Na+ gradient into the cell. In contrast to the facilitated diffusion system for nucleosides, Na(+)-dependent formycin B transport is not inhibited by nitrobenzylthioinosine, but both carrier systems are sensitive to inhibitors of D-glucose transport. Natural purine nucleosides and uridine are strong inhibitors of Na(+)-dependent formycin B transport. Transepithelial flux measurements substantiated that the Na(+)-dependent transport mechanism for formycin B functions as an absorptive system.

Uptake and release of [3H]formycin B via sodium-dependent nucleoside transporters in mouse leukemic L1210/MA27.1 cells.

At least seven functionally distinct nucleoside transport processes exist; however, mouse leukemic L1210/MA27.1 cells possess only one subtype, a Na+-dependent transporter termed N1/cif. The capacity of this transporter subtype to release nucleosides from L1210/MA27.1 cells was investigated with the poorly metabolized inosine analog [3H]formycin B. Uptake of [3H]formycin B into these cells was inhibited by replacement of Na+ in the buffer with choline, or by blocking Na+/K+ ATPase with 2 mM ouabain, inhibiting glycolysis with 5 mM iodoacetic acid or inhibiting nucleoside transport with 1 mM phloridzin. Sodium stimulated uptake with an EC50 value of 12 mM. To measure release of [3H]formycin B, cells were loaded with [3H]formycin B (10 microM) then washed and resuspended in buffer. Replacement of Na+ in the buffer with choline enhanced [3H]formycin B release by 20 to 47%, and significant stimulation of release was observed with Na+ concentrations of 30 mM or less. Resuspending loaded cells into Na+ buffer containing 2 mM ouabain or 10 microM monensin, a Na+ ionophore, significantly enhanced [3H]formycin B release during 20 min by 39% or 29%, respectively. Release of [3H]formycin B into choline buffer was inhibited 26.5% by 10 mM phloridzin and 39.6% by 10 mM propentofylline, compounds known to inhibit various transporters including Na+-dependent nucleoside transporters. Release was also inhibited significantly by 100 microM concentrations of dilazep, dipyridamole and nitrobenzylthioinosine, inhibitors with selectivity for Na+-independent nucleoside transporters. In the absence of Na+, the permeants adenosine and uridine enhanced [3H]formycin B release by up to 40.9% and 21.4%, respectively. These data indicate that in the absence of an inwardly directed Na+ gradient, Na+-dependent nucleoside transporters can function in the release of nucleosides.

Analysis of intestinal perfusion data for highly permeable drugs using a numerical aqueous resistance--nonlinear regression method.

PURPOSE: To develop, validate and apply a method for analyzing the intestinal perfusion data of highly permeable compounds using the Numerical Aqueous Resistance (NAR) theory and nonlinear regression (NAR-NLR) and to compare the results with the well-established Modified Boundary Layer (MBL) Analysis. METHODS: The NAR-NLR method was validated and the results were compared to the MBL analysis results using previously reported cephradine jejunal perfusion data. Using the Single Pass Intestinal Perfusion (SPIP) method, the concentration dependence of intestinal permeability was investigated for formycin B, proline, and thymidine, three compounds reported to be absorbed by carrier-mediated transport processes. The MBL and NAR-NLR analyses were then applied to the three sets of SPIP data. RESULTS: The results demonstrate that the intrinsic MBL transport parameters were highly variable and, in one case, the analyses failed to give a statistically significant Michaelis constant. The MBL mean dimensionless wall permeabilities (P*w) were greater than the NAR-NLR P*w and were also highly variable. In all cases, the NAR-NLR variability was significantly lower than the MBL variability. The extreme variability in the MBL-calculated P*w is due to the sensitivity of P*w when the fraction of unabsorbed drug (Cm/Co) is low or, alternatively, when P*w approached the aqueous permeability, P*aq. CONCLUSIONS: The NAR-NLR method facilitates the analysis of intestinal perfusion data for highly permeable compounds such as those absorbed by carrier-mediated processes at concentrations below their Km. The method also allows for the use of a wider range of flow conditions than the MBL analysis resulting in more reliable and less variable estimates of intestinal transport parameters as well as intestinal wall permeabilities.

Interaction of the novel adenosine uptake inhibitor 3-[1-(6,7-diethoxy-2-morpholinoquinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1H,3H)-quinazolinedione hydrochloride (KF24345) with the es and ei subtypes of equilibrative nucleoside transporters.

Nucleosides such as adenosine, as well as many nucleoside-based drugs, permeate cell membranes via a family of equilibrative nucleoside transporters (ENTs). We assessed the effects of (3-[1-(6,7-diethoxy-2-morpholino-quinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1H,3H)-quinazolinedione hydrochloride (KF24345), a novel anti-inflammatory agent that potentiates the actions of adenosine, on the es (inhibitor-sensitive) and ei (inhibitor-resistant) subtypes of ENTs in human, mouse, and rat cells. KF24345 was similar to the prototypical high-affinity inhibitor nitrobenzylthioinosine (NBMPR) for blocking the human es transporter (K(I) of approximately 0.4 nM), but was 50-fold more effective than NBMPR at blocking the human ei transporter (K(I) of approximately 100 nM). KF24345 displayed significantly less species heterogeneity in its affinity for the es transporter than did dipyridamole, a widely used inhibitor of nucleoside transport; KF24345 may thus prove useful as an inhibitor for studies of nucleoside metabolism in a range of animal models. Furthermore, KF24345 seemed to act as a noncompetitive inhibitor of both [(3)H]NBMPR binding and [(3)H]nucleoside uptake by human es transporters, and these kinetics were consistent with an observed slow dissociation of KF24345 from the inhibitor binding site. KF24345 also exhibited unusual biphasic profiles for inhibition of [(3)H]NBMPR binding to membranes prepared from a recombinant human es transporter model (PK15-hENT1), suggesting the presence of multiple populations of NBMPR binding proteins in these membranes. The atypical tight binding interaction of KF24345 with the es transporter may prove useful for the molecular delineation of inhibitor binding domains and will facilitate its use as an in vivo inhibitor of nucleoside transport in studies focused on the biological effects of adenosine.

Formycin B elimination from the cerebrospinal fluid of the rat.

The goal of this study was to determine whether specific transport systems are involved in nucleoside elimination from the cerebrospinal fluid (CSF). First, in vitro studies were carried out in isolated choroid plexus tissue slices from rat to ascertain the mechanisms of transport of formycin B, a model nucleoside analogue. 3H-Formycin B accumulated against a concentration gradient in the presence of an Na+ gradient in the isolated ATP-depleted choroid plexus tissue slices. This accumulation was reduced by high concentrations of unlabeled formycin B. Nitrobenzylthioinosine (NBMPR), an equilibrative nucleoside transport inhibitor, inhibited the uptake of formycin B in the absence of an Na+ gradient. These data suggest that both equilibrative and secondary active Na(+)-nucleoside transport systems are present in rat choroid plexus. In vivo, formycin B, together with inulin as a bulk flow marker, was injected into the lateral ventricle of the anesthetized rat with the aid of a stereotaxic device, and CSF was sampled from the cisterna magna at various times after injection. Twelve rats were randomized and divided into a low- and a high-dose group. The CSF clearance (CLCSF) of formycin B was significantly higher than the CLCSF of inulin in both animal groups (P < 0.01), indicating that formycin B is cleared from CSF by a pathway(s) in addition to bulk flow. Formycin B CLCSF was significantly lower in the high-dose group than in the low-dose group (P < 0.05), suggesting a saturable CSF elimination. The CLCSF of formycin B was also significantly reduced in animals treated with NBMPR (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Purine uptake and release in rat C6 glioma cells: nucleoside transport and purine metabolism under ATP-depleting conditions.

Adenosine, through activation of membrane-bound receptors, has been reported to have neuroprotective properties during strokes or seizures. The role of astrocytes in regulating brain interstitial adenosine levels has not been clearly defined. We have determined the nucleoside transporters present in rat C6 glioma cells. RT-PCR analysis, (3)H-nucleoside uptake experiments, and [(3)H]nitrobenzylthioinosine ([(3)H]NBMPR) binding assays indicated that the primary functional nucleoside transporter in C6 cells was rENT2, an equilibrative nucleoside transporter (ENT) that is relatively insensitive to inhibition by NBMPR. [(3)H]Formycin B, a poorly metabolized nucleoside analogue, was used to investigate nucleoside release processes, and rENT2 transporters mediated [(3)H]formycin B release from these cells. Adenosine release was investigated by first loading cells with [(3)H]adenine to label adenine nucleotide pools. Tritium release was initiated by inhibiting glycolytic and oxidative ATP generation and thus depleting ATP levels. Our results indicate that during ATP-depleting conditions, AMP catabolism progressed via the reactions AMP --> IMP --> inosine --> hypoxanthine, which accounted for >90% of the evoked tritium release. It was surprising that adenosine was not released during ATP-depleting conditions unless AMP deaminase and adenosine deaminase were inhibited. Inosine release was enhanced by inhibition of purine nucleoside phosphorylase; ENT2 transporters mediated the release of adenosine or inosine. However, inhibition of AMP deaminase/adenosine deaminase or purine nucleoside phosphorylase during ATP depletion produced release of adenosine or inosine, respectively, via the rENT2 transporter. This indicates that C6 glioma cells possess primarily rENT2 nucleoside transporters that function in adenosine uptake but that intracellular metabolism prevents the release of adenosine from these cells even during ATP-depleting conditions.