In vitro and in vivo uroselectivity of B8805-033, an antagonist with high affinity at prostatic alpha1A- vs. alpha1B- and alpha1D-adrenoceptors.

We have investigated the pharmacological properties of B8805-033 [(+/-)- 1,3,5-trimethyl-6-[[3-[4-((2,3-dihydro-2-hydroxymethyl)-1,4-benzodioxin-5-yl)-1-piperazinyl]propyl] amino]-2,4(1H,3H)-pyrimidinedione], a new alpha1A-adrenoceptor (AR) selective antagonist. In radioligand binding studies, B8805-033 was 150- to 1200-fold selective for alpha1A-ARs (pKi rat cerebral cortex 8.70, cloned human receptor 7.71) relative to alpha1B-ARs (pKi rat cerebral cortex 5.60, rat liver 5.39, cloned human receptor 5.16) and alpha1D-ARs (pKi cloned human receptor 5.49). B8805-033 inhibited noradrenaline (NA) induced contractions mediated by alpha1A-ARs in rat vas deferens and rabbit and human prostate (pA2 7.62-8.40) much more potently than those mediated by alpha1B-ARs in guinea pig and mouse spleen or by alpha1D-ARs in rat aorta and pulmonary artery (pA2 5.21-5.52). With the exception of a high agonist affinity at 5-HT1A receptors (pKi 9.74 in pig cortex, pD2 6.82 for contraction of rabbit basilar artery) and a moderate to low affinity at histamine H1-receptors (pA2 6.74) and beta1-ARs (pA2 5.75), B8805-033 did not interact with a number of other neurotransmitter receptors (pKi or pA2<5.0). From the i.v. doses of B8805-033 to either inhibit the urethral pressure response to NA by 50% (29 nmol/kg) or to evoke a fall in diastolic blood pressure by 25% (1.54 micromol/kg) in anaesthetized dogs, an urethral/ vascular selectivity ratio of 52 was obtained, far exceeding that found for the nearly unselective prazosin (ratio 1.8). We conclude that B8805-033 is a highly alpha1A-AR selective antagonist, which may potentially be useful as pharmacological tool to investigate alpha1-AR heterogeneity and in the treatment of benign prostatic hyperplasia.

Pharmacokinetics and drug interactions--relevant factors for the choice of a drug.

Pharmacokinetics serve as a useful tool in drug development by identifying the drug's disposition and elimination characteristics, the absorption characteristics of the biopharmaceutical formulation, and the therapeutic dose regimen in various patient populations. Where two or more drugs of a class have a similar efficacy, the choice of the drug may depend upon the reproducibility of the pharmacokinetics and the minimal risk of drug interaction. Pantoprazole, a selective proton pump inhibitor, appears to meet the above criteria. As opposed to other members of the class, pantoprazole exhibits linear, predictable pharmacokinetics and lack of drug interactions.

Vasodilatation elicited by 5-HT1A receptor agonists in constant-pressure-perfused rat kidney is mediated by blockade of alpha 1A-adrenoceptors.

The vasodilator mechanism of the putative serotonin1A (5-HT) receptor agonists, urapidil, 5-methyl-urapidil, ipsapirone, flesinoxan and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was investigated in constant-pressure perfused rat kidneys. The compounds (10(-12)-10(-7) mol bolus injection) neither enhanced basal flow nor evoked vasodilatation in kidneys preconstricted by 27 mM KCl, 1.5 mM BaCl2 or 10(-6) M prostaglandin (PG)F2 alpha, but evoked a dose-dependent, reversible and spiroxatrine-resistant increase in vasodilatation of organs preconstricted by 6 x 10(-7) M noradrenaline. 5-Carboxamidotryptamine and sumatriptan did not reverse the vasoconstriction induced by all stimuli or that induced by noradrenaline in the presence of 5-HT2 plus 5-HT3 receptor blockade. No correlation for the vasorelaxant drugs was found between their -log ED50 in rat kidney and pKi values at 5-HT1A binding sites in pig cortex as determined in radioligand experiments. The relaxation in rat kidney induced by 5-HT1A receptor agonists and alpha 1A-adrenoceptor-selective antagonists (WB 4101 and (+)-niguldipine) was significantly correlated with pKi values at alpha 1A binding sites in rat cortex and the pA2 values derived from contraction studies for competitive antagonism at alpha 1-adrenoceptors in prostatic portions of the rat vas deferens, but differed from pKi values for alpha 1B binding sites in rat cortex. Thus, the vasodilator effect of the 5-HT1A receptor agonists urapidil, 5-methyl-urapidil, ipsapirone, flesinoxan and 8-OH-DPAT in the noradrenaline-perfused rat kidney appears to be mediated by their concomitant alpha 1A-adrenoceptor blockade. No evidence for a vasodilator effect mediated through 5-HT1A receptors was found under our experimental conditions.

Regional and laminar distributions of alpha 1-adrenoceptors and their subtypes in human and rat hippocampus.

The distributions of the alpha 1-adrenoceptor and its subtypes (alpha 1A and alpha 1B) in human and rat hippocampus are analysed by quantitative receptor autoradiography. alpha 1-Adrenoceptors are labelled by [3H]prazosin. The alpha 1A subtype is visualized by [3H]prazosin after irreversible blockade of alpha 1B adrenoceptors with chloroethylclonidine or directly by [3H]5-methyl-urapidil. The alpha 1B subtype is investigated by [3H]prazosin binding in the presence of the alpha 1A antagonist 5-methyl-urapidil. Considerable differences in the regional and laminar patterns of alpha 1-adrenoceptors are found between rat and human hippocampi. The rat hippocampus is characterized by a low overall density and a rather homogeneous regional and laminar distribution. This is in contrast to the human pattern, which shows a much higher overall level of alpha 1 receptor density and a restriction of alpha 1 receptors to the CA3 region of Ammon's horn and the dentate gyrus. Moreover, alpha 1A and alpha 1B receptors of the human hippocampus are differentially distributed with the alpha 1A subtype concentrated in the hilus and lucidum layer of CA3, and the alpha 1B subtype concentrated in the molecular layer of the dentate gyrus. Additionally, the distribution of alpha 1 receptors is compared with the distribution of 5-hydroxytryptamine 1A receptors. The subtype specific pattern is correlated with the distribution of glutamatergic systems in the human (but not in the rat) hippocampus. alpha 1A Receptor localization coincides with the target area of the mossy fibre system, and alpha 1B receptors are preferentially localized in the target area of the hippocampal associational fibres and partly of the perforant pathway. This result points to possible interactions between noradrenaline- and glutamate-mediated neurotransmission differentiated by topographically segregated alpha 1-adrenoceptor subtypes.

Involvement of 5-HT1A receptors in blood pressure reduction by 8-OH-DPAT and urapidil in cats.

This study investigated the effects of (-)-pindolol, a putative 5-HT1A receptor antagonist, upon the central hypotensive action of the antihypertensive drug urapidil and of the purported 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) in cats. Chloralose/urethane-anesthetized cats were thoracotomized and artificially ventilated. Blood pressure was monitored in the iliac artery, and the drugs were injected into the vertebral artery. Urapidil (1-300 nmol/kg) or 8-OH-DPAT (0.01-1 nmol/kg) dose-dependently reduced blood pressure. (-)-Pindolol (30 and 100 nmol/kg) shifted the dose-response curves of both drugs significantly and in a similar manner to the right. Doses of urapidil of 30 nmol/kg or higher also reduced the elevation of blood pressure following the intravenous injection of the alpha 1-adrenoceptor agonist cirazoline whereas 8-OH-DPAT was ineffective. Yet, the hypotensive response to the directly acting vasodilator nitroglycerin remained unchanged after urapidil. The results support the hypothesis that the centrally mediated component of the antihypertensive action of urapidil is due to stimulation of 5-HT1A receptors in the brainstem. Peripheral alpha 1-adrenoceptor blockade comes into play with higher doses of the drug administered via the vertebral artery.

Involvement of brain 5-HT1A receptors in the hypotensive response to urapidil.

Stimulation of serotonin-1A (5-hydroxytryptamine) (5-HT1A) receptors in the brain stem has been suggested to contribute to the antihypertensive action of the alpha 1-adrenoceptor antagonist urapidil. This hypothesis was tested by analyzing the influence of the 5-HT1A receptor antagonist spiroxatrine on the hypotensive responses to urapidil and the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT). Chloralose/urethane-anesthetized cats underwent thoracotomy and were artificially ventilated. Blood pressure was monitored in the femoral artery. Urapidil (0.01 to 10 mumol/kg) or 8-OH-DPAT (3 to 30 nmol/kg) was injected into a femoral vein and the maximal hypotensive response recorded. A dose-response test with both drugs was performed before and after administration of spiroxatrine (3 and 10 nmol/kg); the latter was given through the vertebral artery, thus delivering the antagonist to the brain stem. Blood pressure was dose-dependently reduced by urapidil and 8-OH-DPAT after intravenous injection. Central administration of spiroxatrine through the vertebral artery shifted the dose-response curves of both drugs markedly and in a dose-dependent manner to the right, while the hypotensive response to the peripheral vasodilator nitroglycerin remained unchanged. The results suggest that the hypotensive response after peripheral administration of urapidil is mediated in part by stimulation of brain 5-HT1A receptors and this effect on central cardiovascular regulation is additive to the blood pressure reduction resulting from peripheral alpha-adrenoceptor blockade.

Evidence for the interaction of urapidil with 5-HT1A receptors in the brain leading to a decrease in blood pressure.

Current knowledge about the role of serotonin (5-HT) in central cardiovascular regulation is reviewed. Results from experiments with the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) suggest that activation of somatodendritic 5-HT1A receptors in the medulla oblongata decreases the firing of serotoninergic neurons and thus reduces their excitatory input to the sympathetic neurons in the intermediolateral cell column. As a consequence, blood pressure is reduced by 5-HT1A receptor agonists. Urapidil is an antihypertensive drug that has a dual mode of action: peripheral alpha-adrenoceptor antagonism and interaction with 5-HT1A receptors in the brain. This profile can adequately explain the vasodilation and lack of significant sympathetic activation observed during urapidil treatment.

Interaction of urapidil with brain serotonin-1A receptors increases the blood pressure reduction due to peripheral alpha-adrenoceptor inhibition.

The alpha-adrenoceptor antagonist urapidil influences central cardiovascular regulation, and this effect is unrelated to alpha-adrenoceptors. Since urapidil has appreciable affinity and selectivity for serotonin-1A (5HT1A) receptors, the activity of urapidil at these sites may be relevant for the centrally mediated component of its antihypertensive action. The latter hypothesis was tested by analysing the influence of the 5HT1A receptor antagonist spiroxatrine on the hypotensive response to urapidil, in comparison with the influence on the hypotensive response to the 5HT1A receptor agonist 8-OH-DPAT (8-hydroxy-2-[di-n-propylamino]tetralin). Anaesthetized cats were thoracotomized and artificially ventilated. Blood pressure was monitored in the descending aorta, and the drugs were injected into the vertebral artery. Spiroxatrine (0.1-3.0 nmol/kg) shifted the cumulative dose response curve (blood pressure reduction) of urapidil (3-20 nmol/kg) and of 8-OH-DPAT (0.01-0.1 nmol/kg) to the right, suggesting competitive antagonism. The results support the hypothesis that the effects of urapidil on central cardiovascular regulation and at least part of the hypotensive effects are due to 5HT1A receptor stimulation.

Urapidil and some analogues with hypotensive properties show high affinities for 5-hydroxytryptamine (5-HT) binding sites of the 5-HT1A subtype and for alpha 1-adrenoceptor binding sites.

The mechanism responsible for the antihypertensive effect of urapidil is not yet completely understood. Its vasodilator action has been attributed to an antagonism at vascular alpha 1-adrenoceptors. However, it has been suggested that a central action contributes to the hypotensive effect. Recently, three potent analogues of urapidil have been described which also lower blood pressure by a central mechanism. 5-Hydroxytryptamine (5-HT) receptors of the 5-HT1A subtype have been implicated with the central control of cardiovascular function. In the present study, the affinities of these urapidil derivatives (5-acetyl, 5-formyl- and 5-methyl-urapidil) for 5-HT receptors were investigated using 3H-8-hydroxy-2-(di-n-propyl-amino)tetralin (3H-8-OH-DPAT), 125I-iodocyanopindolol (125I-ICYP) and 3H-ketanserin for labelling 5-HT1A, 5-HT1B and 5-HT2 binding sites, respectively. 3H-Prazosin and 3H-clonidine were used as selective alpha 1- and alpha 2-adrenoceptor radioligands, respectively. Urapidil and its analogues produced half-maximum inhibition of 3H-8-OH-DPAT binding at concentrations of 4 x 10(-9) mol/l to 4 x 10(-7) mol/l with the following order of potency: urapidil less than 5-acetyl- less than or equal to 5-formyl- less than 5-methyl-urapidil. Thus, 5-methyl-urapidil is one of the most potent ligands at 5-HT1A recognition sites known to date. The IC50 values of urapidil and its derivatives for 3H-prazosin binding were in the range of 5 x 10(-8) mol/l to 8 x 10(-7) mol/l (order of potency: urapidil less than 5-formyl- less than 5-acetyl- less than 5-methyl-urapidil).(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction with histamine H1-receptors and bronchospasmolytic effects of urapidil.

The antihypertensive drug, urapidil, competitively antagonized the histamine-induced contractions in guinea pig isolated tracheal and ileal preparations. Its affinity to histamine H1-receptors was 3-fold higher than that of histamine but 10- and 30-fold weaker than that of diphenhydramine and indoramin, respectively. Urapidil did not inhibit contractions induced by muscarinic agonists in both organs. Investigations in spontaneously breathing guinea pigs showed a greater efficacy of urapidil than that of diphenhydramine in protecting the animals against histamine-induced bronchospasms, whereas acetylcholine-induced spasms were only moderately inhibited. Theophylline, at a 100-fold higher dosage than urapidil, protected the animals against both histamine and acetylcholine challenges. This experimentally observed effect of urapidil supports clinical trials in hypertensive patients suffering also from obstructive lung disease and/or allergic illness.

Influence of urapidil on alpha- and beta-adrenoreceptors in pithed rats.

The interaction of urapidil with pre- and postsynaptic alpha-adrenoreceptors and with postsynaptic beta-adrenoreceptors was studied in pithed normotensive rats and compared to the effects of clonidine, prazosin, and atenolol. I.v. injection of urapidil did not substantially change blood pressure, while clonidine raised blood pressure. Urapidil dose-dependently antagonized the pressor effects of the alpha 1-agonist L-phenylephrine (pDR2 6.8) and of the alpha 2-agonist azepexole (pDR2 5.2). Compared to urapidil, prazosin was a more potent antagonist of phenylephrine at postsynaptic vascular alpha 1-adrenoreceptors (pDR2 8.7) and of azepexole at alpha 2-adrenoreceptors (pDR2 5.6). Urapidil inhibited the tachycardia produced by discontinuous or continuous electrical stimulation of the thoracic spinal outflows (ID50 4.8 and 27.2 mumol/kg, respectively). In contrast to the inhibitory action of clonidine (ID50 0.039 and 0.023 mumol/kg, respectively), the inhibition by urapidil was not reversed by the selective alpha 2-antagonist rauwolscine (10 mumol/kg). Prazosin did not change stimulation-evoked tachycardia but atenolol caused pronounced inhibition (ID50 0.158 mumol/kg, discontinuous stimulation). Urapidil dose-dependently antagonized the tachycardic effect of isoprenaline at beta 1-adrenoreceptors (pDR2 5.1) but also exhibited intrinsic activity by increasing basal heart rate (maximum effect of urapidil was 30% of that of isoprenaline). Urapidil did not change the vasodilatory beta 2-adrenoreceptor-mediated effect of isoprenaline. The results suggest that urapidil is an antagonist at postsynaptic vascular alpha 1- and alpha 2-adrenoreceptors, with a greater potency against alpha 1-adrenoreceptors. An agonistic interaction of urapidil with presynaptic alpha 2-adrenoreceptors could not be demonstrated in pithed rats. Instead, the inhibition by urapidil of stimulation-evoked tachycardia could be accounted for by its beta 1-adrenoreceptor antagonistic effect.

Influence of sulfasalazine, 5-aminosalicylic acid and sulfapyridine on prostanoid synthesis and metabolism in rabbit colonic mucosa.

The effects of sulfasalazine (SASP) and its cleavage products 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP) on prostanoid (PG) synthesis and degradation were determined in rabbit colonic mucosa fractions in vitro. When the microsomal fraction was incubated with (14C)arachidonic acid, 10(-3) M SASP and SP did not markedly change the formation of labeled PGE2, PGF2 alpha, TxB2 and 6-keto-PGF1 alpha X 10(-4) M 5-ASA increased synthesis about 2.7-fold; the pattern of PG identified was unaltered. In the presence of the 10-fold higher concentration of 5-ASA, PG synthesis remained elevated at a similar level. When the cytosolic fraction was incubated with (3H)PGE2, 10(-3) M 5-ASA was without influence and 10(-3) M SP decreased slightly PGE2 breakdown. However, SASP showed a pronounced inhibitory effect at 10(-5) M and inhibition of PGE2 degradation was complete at 10(-3) M SASP. The results are compatible with the assumption that stimulation of PG synthesis by 5-ASA is related to therapeutic benefit in the treatment of ulcerative colitis.

Biotransformation of urapidil: metabolites in serum and urine and their biological activity in vitro and in vivo.

The biotransformation products of the antihypertensive drug urapidil were determined in the serum and urine of rat, dog and man. Comparison of the pattern of urapidil metabolism revealed that all species examined formed the same metabolites. However, quantitative differences were noted. The pattern of metabolites in serum was seen to parallel the excretion of urapidil and its metabolites in urine. The p-hydroxylated urapidil (M1) was found to be the predominant metabolite in man, the uracil-N-demethylated metabolite (M3) in dog, and M1 as well as the O-demethylated urapidil (M2) were found to be important in the rat. Furthermore, trace amounts of the urapidil-N-oxide (M5) are found in dog urine. As determined in the isolated rat vas deferens preparation, urapidil and the synthetized metabolites M1, M2, M3 and M5 are competitive antagonists of the effects of noradrenaline at postsynaptic alpha 1-adrenoceptors. Metabolites M2 and M3 exhibit alpha 1-adrenoceptor antagonism (pA2 values of 6.79 and 6.93 respectively) which are comparable to urapidil (pA2 = 7.02), whilst M1 and M5 are less potent antagonists giving pA2 values of 5.69 and 5.55 respectively. On i.v. or i.j. administration of urapidil, M1, M2, M3 or M5 to anaesthetized, normotensive rats, or orally to conscious spontaneously hypertensive rats, differences in cardiovascular responses were seen. Whilst M1 had little antihypertensive activity, M2 and M3 lowered blood pressure to a similar extent to that seen with urapidil. However, the duration of activity observed was shorter. Urapidil-N-oxide (M5) appeared to possess hypotensive activity comparable to urapidil, but examination of serum samples following the administration of M5 yielded mainly urapidil, suggesting that M5 exhibits its activity following reduction back to urapidil. In view of the serum levels attained and of the biological activity, the metabolites of urapidil do not appear to contribute significantly to the cardiovascular effects of urapidil in man or rat. However, in view of the serum levels and duration of M3 observed in dogs, this metabolite may make a significant contribution to the hypotensive activity of urapidil in this species.

Theoretical analysis of adenosine release from cardiac tissue as influenced by transport inhibitors.

The release of adenosine from cardiac tissue was simulated by use of a model equation which consists of a saturable transfer term for both unidirectional influx and efflux, representing a symmetrical facilitated diffusion mechanism. This proposed model can account for positive and negative changes in adenosine release from cardiac tissue brought about by competitive transport inhibitors.

Metabolism and disposition of 2',3'-di-O-nitro-adenosine-5'-(N-ethyl-carboxamide) in dogs.

2',3'-Di-O-nitro-[8-3H]-adenosine-5'-(N-ethyl-carboxamide) (20 micrograms/kg) was denitrated completely within 1-3 hr in perorally and intravenously dosed dogs. Extremely rapid disappearance of the unchanged drug in serum was parallelled by the instantaneous appearance of mononitrates with 3'-mononitrate levels exceeding those of 2'-mononitrate three-fold. The mononitrates were eliminated with a half-life of 30-70 min, giving rise to the completely denitrated product, adenosine-5'-(N-ethyl-carboxamide) (NECA). The latter product was not further metabolized and was eliminated with a half-life of about 4 hr. Urinary excretion averaged 50% of the administered dose within 4 days and was represented essentially by the completely denitrated drug. Volatile 3H-label of the drug was found in serum and urine during in vivo experiments. Oral bioavailability of the drug was about 90%. In vitro studies indicated that thiols are involved in denitration and reactions are catalysed by glutathione S-transferases, which were partially purified from dog liver. Nitrate ester cleavage was more easily accomplished at the 2'-position than at the 3'-position of the drug and resulted in the liberation of inorganic nitrite. Comparison of in vitro denitration rates gave the following ranking order; 2',3'-di-O-nitro-NECA greater than isosorbide-2,5-dinitrate greater than 2'-nitro-NECA greater than 3'-nitro-NECA greater than isosorbide-2-mononitrate, while nitrate ester cleavage of isosorbide-5-mononitrate was not detectable.

Modification by nitrobenzylthioinosine-5'-monophosphate of pseudoisocytidine pharmacokinetics in mice and rats through inhibition of membrane transport.

In isolated, perfused mouse livers, initial rates of uptake of [2-14C]pseudoisocytidine (PIC), measured during the first 15 secs of perfusion were markedly reduced when the perfusion medium contained 5 X 10(-6) M nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport. A similar inhibition of PIC uptake occurred when mice were treated with NBMPR-P (the 5'-monophosphate of NBMPR) at doses greater than 0.2 mg/kg ip injected 30 mins prior to the liver perfusion assay. However, in vivo studies showed that a late effect of NBMPR-P was enhancement in PIC levels in liver and other tissues in mice and rats, relative to levels in animals that had not received NBMPR-P. Increases in incorporation of PIC into RNA reflected the NBMPR-P-induced increases in tissue levels of PIC. NBMPR-P and other inhibitors of nucleoside transport may have therapeutic applications in manipulation of the pharmacokinetic behavior and toxicity of nucleoside drugs.

Manipulation of toxicity and tissue distribution of tubercidin in mice by nitrobenzylthioinosine 5'-monophosphate.

The i.v. administration of tubercidin, an analog of adenosine, in a single dose of 45 mg/kg caused death in about 90% of B10D2F1 mice so treated. Serum and urine analysis, as well as histological examination of tissues, related the lethality of tubercidin to hepatic injury, which was markedly reduced when mice were treated with the inhibitor of nucleoside transport, nitrobenzylthioinosine 5'-monophosphate (NBMPR-P), at i.p. doses higher than 10 mg/kg 30 min prior to tubercidin injection. With high NBMPR-P doses (100 mg/kg, i.p.) followed by tubercidin injection (45 mg/kg, i.v.), kidney damage and high mortality occurred. The tissue distribution of 3H following (( G-3H]tubercidin administration paralleled hepatic or renal injury: NBMPR-P treatment decreased the content of tubercidin-derived 3H in liver and increased that in kidney. Furthermore, the half-life of the decline in tubercidin levels in serum during the first minute after[3H]tubercidin administration was longer in NBMPR-P-treated mice (26 sec) than in untreated mice (10 sec), with the result that 3H levels in serum were more than ten times higher in the former than in the latter at an early stage during the distribution of tubercidin. Within 15 min after i.p. administration, the tissue distribution of (( 3H]tubercidin was complete. The i.p. administration of tubercidin caused ascites and the appearance of amylase in the peritoneal fluid evidently because of peritonitis and pancreatic injury. Administration of NBMPR-P by the i.p. route, but not by the i.v. route, prevented these injuries and shifted the LD50 of i.p. injected tubercidin (5 mg/kg) to markedly higher values (a 4-fold increase with NBMPR-P at 100 mg/kg). The protection of mice by NBMPR-P against lethal injuries caused by i.p. injected tubercidin was consistent with the inhibition by NBMPR-P of tubercidin accumulation in mesentery and pancreas. The tissue specificity of the NBMPR-P influence on the tissue distribution of tubercidin may reflect differences in NBMPR-P pharmacokinetics and/or in properties of the nucleoside permeation mechanism among various tissues.

Uptake of cytidine by isolated, perfused mouse liver.

Mouse livers were perfused at 22 degrees C with an oxygenated salts medium containing [5-3H]cytidine and [carboxyl-14C]inulin. The cellular uptake of cytidine was determined from the 3H content of liver samples less that present in the extracellular (inulin) space of the samples. Time courses of cytidine uptake were biphasic with initial phases which were approximately linear for 15 s and had time zero values that approximated the extracellular space. Rates of cytidine uptake derived from the initial phase of uptake evidently represented rates of membrane transport because (i) initial rates were saturable, and (ii) cytidine uptake was blocked by the nucleoside transport inhibitor, nitrobenzylthioinosine (NBMPR). Treatment of mice with the 5'-monophosphate of NBMPR (greater than 0.2 mg/kg, injected i.p.) 30-40 min prior to the perfusion also blocked cytidine entry into livers. An apparent half-saturation constant of about 10(-3) M and a maximum rate of about 1 mumol . g-1 . min-1 were estimated for the NBMPR-sensitive transport of cytidine into mouse liver cells.

Absence of binding sites for the transport inhibitor nitrobenzylthioinosine on nucleoside transport-deficient mouse lymphoma cells.

Cells of an adenosine-resistant clone (AE1) of S49 mouse lymphoma cells were compared with cells of the parental line with respect to (a) characteristics of nucleoside transport, (b) high affinity binding of the inhibitor of nucleoside transport, nitrobenzylthioinosine (NBMPR), and (c) the antiproliferative effects of the nucleoside antibiotics, tubercidin, arabinosyladenine and showdomycin. Rates of inward transport of uridine, thymidine, adenosine, 2'-deoxyadenosine, tubercidin, showdomycin, and arabinosyladenine in AE1 cells were less than 1% of those in cells of the parental S49 line. The inhibitor of nucleoside transport, NBMPR, reduced rates of inward nucleoside transport in S49 cells to levels comparable to those seen in the transport-defective mutant. S49 cells possessed high affinity sites that bound NBMPR (6.6 X 10(4) sites/cell, Kd = 0.2 nM), whereas site-specific binding of NBMPR to AE1 cells was not demonstrable, indicating that loss of nucleoside transport activity in AE1 cells was accompanied by loss of the high affinity NBMPR binding sites. Relative to S49 cells, AE1 cells were resistant to the antiproliferative effects of tubercidin and showdomycin, but differences between the two cell lines in sensitivity toward arabinosyladenine were minor, suggesting that nucleoside transport activity was required for cytotoxicity of tubercidin and showdomycin, but not for that of arabinosyladenine.