Similarities between cytomegalovirus-induced cell rounding and contraction of smooth muscle cells

Cytomegalovirus infection of human fibroblastic cell cultures resulted in rounding of the cells and a subsequent decrease in their size. Smooth muscle relaxing agents such as papaverine, hydralazine, diazoxide, nitroprusside and verapamil blocked these early cytopathogenic effects suggesting that cell rounding results from a contractile-like response involving an influx of calcium ions.     

Amiloride inhibition of human cytomegalovirus replication.

Amiloride, an inhibitor of Na+/H+ exchange, interfered with cytomegalovirus (CMV) DNA synthesis, blocked the formation of nuclear inclusions, and reduced CMV infectious yields. The reduction of CMV infectious yields was concentration dependent with an ED90 of 46 microM. Amiloride at a concentration of 150 microM reduced CMV yields by about 100-fold. Reduction of infectious yields appeared to be related to interference with the formation of nuclear inclusions and to inhibition of CMV DNA synthesis. Nuclear inclusions were much reduced in size and demonstrated poorly defined cellulae in the amiloride-treated cells. CMV DNA synthesis was inhibited by approximately 70% when cells were treated with 150 microM amiloride. The reduction in CMV yields could not be related to the reported inhibitory effect of amiloride on protein synthesis. In amiloride (150 microM)-treated, CMV-infected cells, late, yet not immediate-early or early, protein synthesis was markedly decreased relative to untreated, CMV-infected cells. Accordingly, CMV DNA synthesis and the replication of CMV may be related to Na+ entry through an amiloride-sensitive pathway.     

Direct evidence for the interaction between papaverine and alpha 2-adrenergic receptors in canine platelets

The effects of papaverine on specific [3H]-yohimbine binding to canine platelet alpha 2-adrenergic receptors and on the platelet aggregation were assessed and compared with those of verapamil. Both compounds concentration-dependently inhibited [3H]-yohimbine binding with KI values for respective compounds of 0.39 +/- 0.05 microM (n = 3) and 15 +/- 0.19 microM (n = 3). In the presence of either compound KD values in Scatchard analysis of the equilibrium ligand binding increased in concentration-dependent manner, whereas Bmax did not change, indicating competitive inhibition of the ligand binding by these compounds. (-)-Epinephrine (3 microM) potentiation of adenosine diphosphate (ADP, 0.1 microM) aggregation was inhibited by papaverine with IC50 of 11 +/- 3.6 microM (n = 4). In the same experiments verapamil inhibited the platelet aggregation with lower IC50 (3.1 +/- 0.87 microM, n = 4) in comparison with that for papaverine. These results suggest that papaverine, like verapamil, inhibits physiological response of canine platelets through alpha-adrenergic receptor stimulation by direct interaction with the receptors.     

A mechanism for P-glycoprotein-mediated apoptosis as revealed by verapamil hypersensitivity

Selection of tumor cell lines with anticancer drugs has led to the appearance of multidrug-resistant (MDR) subclones with P-glycoprotein 1 (P-gp1) expression. These cells are cross-resistant to several structurally and functionally dissimilar drugs. Interestingly, in the process of gaining resistance, MDR cells become hypersensitive or collaterally sensitive to membrane-active agents, such as calcium channel blockers, steroids, and local anaesthetics. In this report, hypersensitivity to the calcium channel blocker, verapamil, was analyzed in sensitive and resistant CHO cell lines. Our results show that treatment with verapamil preferentially induced apoptosis in MDR cells compared to drug-sensitive cells. This effect was independent of p53 activity and could be inhibited by overexpression of the Bcl-2 gene. The induction of apoptosis by verapamil had a biphasic trend in which maximum cell death occurred at 10 microM, followed by improved cell survival at higher concentrations (50 microM). We correlated this effect to a similar biphasic trend in P-gp1 ATPase activation by verapamil in which low concentrations of verapamil (10 microM) activated ATPase, followed by inhibition at higher concentrations. To confirm the relationship between apoptosis and ATPase activity, we used two inhibitors of P-gp1 ATPase, PSC 833 and ivermectin. These ATPase inhibitors reduced hypersensitivity to verapamil in MDR cells. In addition, low concentrations of verapamil resulted in the production of reactive oxygen species (ROS) in MDR cells. Taken together, these results show that apoptosis was preferentially induced by P-gp1 expressing cells exposed to verapamil, an effect that was mediated by ROS, produced in response the high ATP demand by P-gp1.     

N omega-nitro-L-arginine: a potent inhibitor of the L-arginine-dependent soluble guanylate cyclase activation pathway in LLC-PK1 cells

Oxytocin increased cyclic GMP levels in LLC-PK1 porcine kidney epithelial cells through activation of soluble guanylate cyclase. NG-Monomethyl-L-arginine and N omega-nitro-L-arginine inhibited oxytocin (10 microM) induced cyclic GMP accumulation with IC50 values of 2.3 microM and 140 nM, respectively, and the inhibition was prevented with L-arginine. Both inhibitors at 100 microM lowered the basal levels of cyclic GMP, but did not affect those induced by 1 microM sodium nitroprusside and 100 nM atrial natriuretic factor. These data support our hypothesis that an endothelium-derived relaxing factor-like substance is formed as the endogenous activator of soluble guanylate cyclase in an L-arginine-dependent fashion in various cell types. N omega-Nitro-L-arginine is 16 times more potent than NG-monomethyl-L-arginine as a specific inhibitor of this pathway in LLC-PK1 cells.     

Decreased cytomegalovirus expression following proinflammatory cytokine treatment of primary human astrocytes

Understanding the influence of immune effector mechanisms on CMV infection of the CNS may facilitate the development of immunotherapies for viral encephalitis. Using cultures of highly purified, fully permissive primary human astrocytes, proinflammatory cytokines, but not antiinflammatory cytokines or beta-chemokines, were found to inhibit CMV expression, DNA synthesis, and replication. Treatment with certain proinflammatory cytokines 24 h before CMV infection markedly suppressed viral expression in astrocytes. TNF-alpha, IL-1beta, and IFN-gamma all inhibited CMV expression (70 +/- 4.2%, 65 +/- 3.4%, and 82 +/- 3.6% inhibition of viral expression, respectively, n = 5). In contrast, no viral suppression was observed following IL-6 treatment. Suppressive activity was dependent on the addition of cytokines before CMV infection. Cytokine pretreatment did not affect CMV entry into primary astrocytes, and the observed cytokine-induced suppressive activity was not affected by the NO synthase inhibitor NG-monomethyl- -arginine (NGMA). Instead, the suppressive effect appeared to be mediated through a mechanism involving inhibition of CMV major immediate early promoter activity. These results support the hypothesis that proinflammatory cytokines possess anti-CMV activity in brain cells and may lead to new interventions for CMV encephalitis based upon immunotherapy.     

Effect of verapamil on glycogenolysis and gluconeogenesis in the perfused rat liver

In perfused livers from fed rats, rates of glucose production (glycogenolysis) were 133 +/- 12 mumol/g/hr. Infusion of 2 microM verapamil into these livers decreased the rates of glucose production significantly to 97 +/- 15 mumol/g/hr within 10 min. Conversely, rates of production of lactate plus pyruvate (glycolysis) of 64 +/- 6 mumol/g/hr were not significantly altered by verapamil (60 +/- 3 mumol/g/hr). When 50 microM verapamil was infused, however, rates of both glycogenolysis and glycolysis were diminished to 56 +/- 11 and 43 +/- 5 mumol/g/hr, respectively. In perfused livers from fasted rats, infusion of 20 mM fructose increased the rates of production of glucose (gluconeogenesis) significantly from 11 +/- 7 to 121 +/- 17 mumol/g/hr. These rates reached 138 +/- 7 mumol/g/hr upon the simultaneous infusion of verapamil (2 microM). In these livers, fructose also increased rates of production of lactate from 6 +/- 2 to 132 +/- 11 mumol/g/hr, which were further increased to 143 +/- 8 mumol/g/hr when 2 microM verapamil was infused. The results show that calcium-dependent processes involved in hepatic carbohydrate metabolism respond differently to the calcium channel blocker verapamil. Low concentrations of verapamil inhibited glycogenolysis significantly while having no effect on either glycolysis or gluconeogenesis. These data suggest that these two processes have different sensitivities to changes in intracellular calcium concentrations and/or different sources of regulatory calcium.     

D,L-alpha-difluoromethylornithine inhibits human cytomegalovirus replication.

D,L-alpha-Difluoromethylornithine (DFMO) is an inhibitor of ornithine decarboxylase, the first enzyme in the polyamine biosynthetic pathway. Exposure of human foreskin fibroblast cells to DFMO before their infection with human strains of cytomegalovirus (CMV) resulted in a reduction in the amount of infectious virus produced. A 3-day exposure to the drug was required to elicit maximal antiviral effect. Cells exposed to DFMO at the time of infection produced normal amounts of infectious virus. Preexposure to the drug for 1, 2, or 3 days before infection resulted in at least 10-, 100-, or 1,000-fold decreases, respectively, in the amount of infectious virus produced. This decrease paralleled the loss of intracellular spermidine and was partially spared by the addition of exogenous putrescine, spermidine, or spermine (10 microM). When added 3 days before infection, DFMO depressed production of herpes simplex virus and simian CMV, as well as wild-type and laboratory prototype strains of human CMV. Although some antiviral effect was observed at a drug concentration of 1 mM, 10 mM gave a stronger effect and was the amount routinely used. At 30 mM DFMO, growth of noninfected cells was slowed but not arrested. Studies to investigate the level at which DFMO interferes with CMV replication showed that DFMO-treated, infected cells (i) exhibit a typical CMV-specific cytopathic effect, (ii) synthesize both viral proteins and viral DNA, (iii) contain at least some capsid forms, and (iv) shed greatly reduced amounts of virus particles into the growth medium. Since CMV virions, like those of herpes simplex virus, contain the polyamines spermidine and spermine, and since DFMO essentially eliminates the pool of intracellular spermidine, the possibility is suggested that this drug may exert its antiviral effect by interfering with virus assembly, perhaps at the level of DNA packaging or capsid envelopment or both.     

Dietary copper deficiency and endothelium-dependent relaxation of rat aorta.

Endothelium-dependent relaxation of aortas was studied in dietary copper (Cu) deficiency. Male, weanling Sprague-Dawley rats were fed diets deficient (CuD, less than 0.5 ppm) or adequate (CuA, 5.0-5.5 ppm) in Cu for 4 weeks. Aortic rings from paired Cu-deficient and Cu-adequate rats were isolated from the descending thoracic aorta, placed in tandem tissue baths, and attached to force transducers. Aortas were contracted with phenylephrine (3 x 10(-7) M) and the degree of force reduction was measured after successively increasing the dose of acetylcholine (10(-8)-10(-5) M), histamine 10(-6)-10(-3) M), or sodium nitroprusside (10(-9)-10(-6) M). Cu deficiency was found to significantly reduce the relaxation responses of each relaxing agent at the highest three of the four doses tested. The ability of Cu-adequate and Cu-deficient aortas to relax was not different, as indicated by their complete relaxation in response to 10(-4) or 10(-5) M papaverine. Because the relaxation responses to both acetylcholine and histamine in rat aorta are dependent on the presence of endothelium, the reduction of these responses suggests that endothelium, or its interaction with smooth muscle, was disrupted in dietary Cu deficiency. The reduction in response to sodium nitroprusside, an endothelium-independent analog of endothelium-derived relaxing factor, indicates that the interaction of endothelium-derived relaxing factor with smooth muscle was disrupted. These findings have implications regarding blood pressure regulation in Cu deficiency.     

Polyamine-mediated inhibition of in-vitro cell proliferation is not due to acrolein

The influence of acrolein or spermine on the viability and growth of phytohaemagglutinin-stimulated rat thymic lymphocytes in cultures supplemented with foetal calf serum have been investigated. Acrolein (greater than 20 microM) was cytotoxic; spermine had little effect on viability, but inhibited [3H]TdR incorporation at low concentrations (approximately 10 microM). Cells treated with greater than 8 microM acrolein 3 hr before stimulation exhibited irreversible inhibition of protein synthesis, whereas 50 microM spermine had no effect, even when cells were treated for 24 hr before stimulation. However, addition of 25 microM spermine after stimulation did inhibit both [3H]-uridine incorporation and protein synthesis: this was reversible if cells were freed of polyamine within 4 hr, but not if washed after 24 hr. These results show that, contrary to several previous reports, in-vitro inhibition of cell proliferation by spermine is not due to the formation and action of acrolein.     

Inhibition of the acetylcholine-induced relaxation of canine isolated basilar artery by potassium-conductance blockers

Canine basilar artery rings precontracted with 5-hydroxytryptamine (0.1-0.5 microM) relaxed in the presence of acetylcholine (25-100 microM), sodium nitroprusside (0.1 microM), or stimulation of the electrogenic sodium pump by restoration of extracellular K+ (4.5 mM) after K(+)-deprivation. Acetylcholine-induced relaxation is believed to be caused by the release of endothelium-derived relaxing factor (EDRF) and is prevented by mechanical removal of the endothelium, while relaxations induced by sodium nitroprusside or restarting of the sodium pump are endothelium-independent. Acetylcholine-induced relaxation was selectively blocked by pretreatment of the tissue with the nonselective K+ conductance inhibitors, 4-aminopyridine (4-AP, 3 mM), Ba2+ (1 mM), and tetraethylammonium (20 mM), 4-AP also blocked ACh-mediated relaxation in muscles contracted with elevated external K+. Relaxation of 5-hydroxytryptamine-induced contraction by sodium nitroprusside, or by addition of K+ to K(+)-deprived muscle, was not affected by 4-AP. Relaxation of basilar artery with acidified sodium nitrite solution (containing nitric oxide) was reduced by 4-AP. These results suggest that 4-AP and possibly Ba2+ inhibit acetylcholine-induced endothelium-dependent relaxation by inhibition of the action of EDRF on the smooth muscle rather than through inhibition of release of EDRF. The increase in K+ conductance involved in acetylcholine-induced relaxation is not due to ATP-inhibited K+ channels, as it is not blocked by glyburide (10(-6) M). Endothelium-derived relaxant factor(s) may relax smooth muscle by mode(s) of action different from that of sodium nitroprusside or by hyperpolarization due to the electrogenic sodium pumping.(ABSTRACT TRUNCATED AT 250 WORDS)     

A single-step silver enhancement method permitting rapid diagnosis of cytomegalovirus infection in formalin-fixed, paraffin-embedded tissue sections by in situ hybridization and immunoperoxidase detection

A single-step silver enhancement method was developed which intensifies the polymerized nickel-complexed diaminobenzidine (Ni-DAB) reaction product of peroxidase. With such enhancement, an in situ hybridization procedure can be performed in less than 8 hr by using a 2-hr hybridization incubation and direct detection. Cytomegalovirus (CMV)-infected lung sections were hybridized in situ for 2 hr with a biotinylated CMV genomic-length probe. The probe was detected directly with avidin-biotinylated peroxidase using Ni-DAB as the substrate, and the reaction product was enhanced with silver. Silver was deposited only on the Ni-DAB and not on normally argyrophilic substances. Indirect detection of the probe using a sandwich technique before silver enhancement proved more sensitive, but the length of the procedure was increased without substantially changing the result (infection vs. no infection). Sensitivity was also improved by omitting the dehydration step before applying the probe, and by increasing the temperature and duration of denaturation. In a blinded study of 21 open-lung biopsies, 13 of 13 culture-negative specimens were negative by hybridization, and 7 of 8 culture-positive specimens were positive by hybridization. Modified short hybridization with a biotinylated probe and silver-enhanced direct detection therefore provides a rapid but sensitive method for diagnosis of viral infection.     

Artemisinin-Derived Dimers Have Greatly Improved Anti-Cytomegalovirus Activity Compared to Artemisinin Monomers

Background

Artesunate, an artemisinin-derived monomer, was reported to inhibit Cytomegalovirus (CMV) replication. We aimed to compare the in-vitro anti-CMV activity of several artemisinin-derived monomers and newly synthesized artemisinin dimers.

Methods

Four artemisinin monomers and two novel artemisinin-derived dimers were tested for anti-CMV activity in human fibroblasts infected with luciferase-tagged highly–passaged laboratory adapted strain (Towne), and a clinical CMV isolate. Compounds were evaluated for CMV inhibition and cytotoxicity.

Results

Artemisinin dimers effectively inhibited CMV replication in human foreskin fibroblasts and human embryonic lung fibroblasts (EC₅₀ for dimer sulfone carbamate and dimer primary alcohol 0.06±0.00 µM and 0.15±0.02 µM respectively, in human foreskin fibroblasts) with no cytotxicity at concentrations required for complete CMV inhibition. All four artemisinin monomers (artemisinin, artesunate, artemether and artefanilide) shared a similar degree of CMV inhibition amongst themselves (in µM concentrations) which was significantly less than the inhibition achieved with artemisinin dimers (P<0.0001). Similar to monomers, inhibition of CMV with artemisinin dimers appeared early in the virus life cycle as reflected by decreased expression of the immediate early (IE1) protein.

Conclusions

Artemisinin dimers are potent and non-cytotoxic inhibitors of CMV replication. These compounds should be studied as potential therapeutic agents for the treatment of CMV infection in humans.     

Clinical potency of calcium channel blockers in asthma may be related to their inhibition of receptor-mediated phasic responses in vitro.

The calcium channel blockers (CCB) have been clinically effective in exercise-induced asthma. The completeness of protection with the CCB might be related specifically to inhibition of Ca2+ influx or release. To examine this hypothesis, the rank order of potency of inhibition of the CCB, nicardipine, diltiazem and verapamil on the steady-state and kinetic parameters of the phasic and tonic responses to the muscarinic receptor agonist carbachol (10 microM) and KCl (40 mM) in the intact isolated guinea-pig trachea was determined. The Ca2+ channel agonist Bay K 8644 was also examined for its effects on intracellular Ca2+. Nicardipine abolished the KCl response at both 0.1 microM and 1 microM concentrations. The amplitude of the KCl response was inhibited equally by 1 microM diltiazem (61% inhibition) and 1 microM verapamil (68% inhibition). The rate constant of onset of the KCl response was similarly inhibited 60% by diltiazem and 66% by verapamil. Nicardipine abolished the carbachol phasic response at the 1 microM concentration. The amplitude of the phasic response was inhibited equally by 0.1 microM nicardipine (61.3% inhibition), 1 microM diltiazem (64.5% inhibition) and 1 microM verapamil (71% inhibition). The rate constant of decay of the phasic response was inhibited equally by 0.1 microM nicardipine (43% inhibition) and 1 microM diltiazem (29% inhibition). The rate constant of onset of the phasic response was unaffected by nicardipine, diltiazem and verapamil. Only 1 microM nicardipine inhibited the amplitude and rate constant of onset of the tonic response. The only effect of Bay K 8644 (1 microM) was to increase the phasic response amplitude. The CCB demonstrate a similar order of potency for inhibition of the phasic responses and clinical efficacy of the CCB in exercise-induced asthma (nicardipine > verapamil > diltiazem).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of the antimicrobial peptide, D-hecate, on trichomonads

Tritrichomonas foetus and Trichomonas vaginalis are protozoan parasites that cause sexually transmitted diseases in cattle and humans, respectively. There is a need for new antimicrobial agents to treat or prevent trichomoniasis because there are currently no approved chemotherapeutic agents against T. foetus and resistance of T. vaginalis to metronidazole does occur. Therefore, we evaluated the effect of a novel antimicrobial peptide, D-hecate, on the viability of 6 isolates of T. foetus and T. vaginalis in vitro. Tritrichomonas foetus and T. vaginalis were grown to mid log phase (24 hr) or late log/stationary phase (48 hr). Parasites at 10(6)/ml were mixed with equal volumes of D-hecate to final concentrations of 10 microM, 20 microM. and 40 microM of D-hecate. Controls had minimal essential medium (MEM) alone. The numbers of viable parasites were determined microscopically after 10, 20, and 30 min of incubation at 37 C with D-hecate or MEM. Our results show that D-hecate killed all 6 isolates of T. foetus and T. vaginalis evaluated. The killing effect was dependent on the concentration of the peptide, incubation time, and phase of growth of the parasites. Ultrastructural studies of parasites treated with 10 microM of D-hecate revealed extensive damage to the plasma membrane of most T. foetus and T. vaginalis cells, while a few cells were distorted but remained intact. D-Hecate may be a useful chemotherapeutic agent for the treatment of trichomoniasis.     

Synthesis of Ribonucleic Acid in Cells Infected with LSc Poliovirus at Elevated Temperatures

The synthesis of viral ribonucleic acid (RNA) was detected within 2 hr after infection with LSc poliovirus at 35 C. This RNA eluted as a single peak with 0.9 m NaCl on methylated albumin celite columns, was sensitive to ribonuclease, precipitated in the presence of 2 m LiCl, and had an S(20) value at 34 +/- 2 in linear sucrose gradients. When cells were infected at 39 to 40 C, there was also early synthesis of RNA. However, 2 hr after infection this synthesis was drastically inhibited. The absence of net RNA synthesis at 39 to 40 C during the late stages of infection was not caused by rapid degradation of newly formed RNA, since the RNA produced between 1 and 2 hr at 39 to 40 C was still present 3.5 hr after infection. There was a 3 log(10) inhibition in the production of infectious virus when p-fluorophenylalanine was present in the medium at a concentration of 25 mug/ml. This concentration of analogue had little effect upon the production of viral polymerase and viral RNA. Virus grown in the presence of analogue at a concentration of 10 mug/ml exhibited increased heat sensitivity compared to control virus. However, viral polymerase exhibited no change in sensitivity to heat or manganese when cells were exposed to 25 mug of p-fluorophenylalanine per ml during infection. p-Fluorophenylalanine had a relatively selective effect on viral capsid protein but did not reverse the inhibition of synthesis of viral RNA at 39 to 40 C.     

Cyclosporine inhibits mouse cytomegalovirus infection via a cyclophilin-dependent pathway specifically in neural stem/progenitor cells

The potential of neural stem and progenitor cell (NSPC) transplantation in neurodegenerative disease raises a concern about immunosuppressive agents and opportunistic neurotropic pathogens that may interfere with engraftment. Cytomegalovirus (CMV) is an important opportunistic pathogen infecting the central nervous system, where it may remain latent for life, following transplacental transmission. Cyclosporine (Cs), an immunosuppressive drug used in organ transplantation, where its use is associated with CMV reactivation, suppressed murine CMV (MCMV) infection in cultured NSPCs but not in fibroblasts. This activity of Cs appears to be mediated via cyclophilin (CyP) rather than via calcineurin. First, the calcineurin-specific inhibitor FK506 failed to suppress replication. Second, the CyP-specific inhibitor NIM811 strongly suppressed replication in NSPC. NSPCs maintained in the presence of NIM811 retained viral genomes for several weeks without detectable viral gene expression or obvious deleterious effects. The withdrawal of NIM811 reactivated viral replication, suggesting that the inhibitory mechanism was reversible. Finally, inhibition of endogenous CyP A (CyPA) by small interfering RNA also inhibited replication in NSPCs. These results show that MCMV replication depends upon cellular CyPA pathways in NSPCs (in a specific cell type-dependent fashion), that CyPA plays an important role in viral infection in this cell type, and that inhibition of viral replication via CyP leads to persistence of the viral genome without cell damage. Further, the calcineurin-signaling pathway conferring immunosuppression in T cells does not influence viral replication in a detectable fashion.     

Nucleoside transport in cultured mammalian cells. Multiple forms with different sensitivity to inhibition by nitrobenzylthioinosine or hypoxanthine

The zero-trans influx of 500 microM uridine by CHO, P388, L1210 and L929 cells was inhibited by nitrobenzylthioinosine ( NBTI ) in a biphasic manner; 60-70% of total uridine influx by CHO cells and about 90% of that in P388, L1210 and L929 cells was inhibited by nmolar concentrations of NBTI (ID50 = 3-10 nM) and is designated NBTI -sensitive transport. The residual transport activity, designated NBTI -resistant transport, was inhibited by NBTI only at concentrations above 1 microM (ID50 = 10-50 microM). S49 cells exhibited only NBTI -sensitive uridine transport, whereas Novikoff cells exhibited only NBTI -resistant uridine transport. In all instances NBTI -sensitive transport correlated with the presence of between 7 7 X 10(4) and 7 X 10(5) high-affinity NBTI binding sites/cell (Kd = 0.3-1 nM). Novikoff cells lacked such sites. The two types of nucleoside transport, NBTI -resistant and NBTI -sensitive, were indistinguishable in substrate affinity, temperature dependence, substrate specificity, inhibition by structurally unrelated substances, such as dipyridamole or papaverine, and inhibition by sulfhydryl reagents or hypoxanthine. We suggest, therefore, that a single nucleoside transporter can exist in an NBTI -sensitive and an NBTI -resistant form depending on its disposition in the plasma membrane. The sensitive form expresses a high-affinity NBTI binding site(s) which is probably made up of the substrate binding site plus a hydrophobic region which interacts with the lipophilic nitrobenzyl group of NBTI . The latter site seems to be unavailable in NBTI -resistant transporters. The proportion of NBTI -resistant and sensitive uridine transport was constant during proportion of NBTI -resistant and sensitive uridine transport was constant during progression of P388 cells through the cell cycle and independent of the growth stage of the cells in culture. There were additional differences in uridine transport between cell lines which, however, did not correlate with NBTI sensitivity and might be related to the species origin of the cells. Uridine transport in Novikoff cells was more sensitive to inhibition by dipyridamole and papaverine than that in all other cell lines tested, whereas uridine transport in CHO cells was the most sensitive to inactivation by sulfhydryl reagents.