Cycloheximide and passive avoidance memory in mice: Time-response,dose-response and short-term memory

The greatest loss of memory shown by mice 24 hr after learning was found to occur with cycloheximide (CXM) (120 mg/kg) administered subcutaneously 30 min before training. With injection at this time the extent of the amnesia was done dependent (30–150 mg/kg) and the resultant amnesia was found to be relatively constant when tested at 1, 7 or 14 days. An attempt was made to follow the development of this amnesia with 100 and 120 mg/kg CXM. However, the saline controls showed an unexpectedly low avoidance 6 hr after training. This was interpreted as a possible interaction between the stress of the injection and the 6 hr interval. An experiment designed to test this possibility showed that mice injected with 0.1 ml of 1% lignocaine gave high avoidance at 6 hr but mice receiving only a needle puncture of the skin gave performances similar to mice receiving saline injections. It was felt that these findings cast doubt on the usefulness of the passive avoidance task in the assessment of drug action on short term memory.     

Behavioral and biochemical effects of the carbamate insecticide,MOBAM

Decreases in rat plasma, erythrocyte and brain cholinesterase levels after intraperitoneal injection of 1 to 5 mg/kg of 4-benzothienyl-N-methylcarbamate (MOBAM) were compared with decrements in both spontaneous motor activity and conditioned avoidance performance produced by this compound. Significant effects were observed with all five measured phenomena at dosages producing no obvious clinical signs. In albino rats, a dosage of 2 mg/kg significantly depressed plasma and erythrocyte cholinesterase activity, and decreased motor activity 15 min after injection but only higher dosages (3 and 5 mg/kg) significantly depressed brain cholinesterase activity and avoidance performance. In Long-Evans rats, both brain cholinesterase activity and avoidance performance were significantly reduced by the lower (2 mg/kg) dosage. The avoidance impairments observed after 3 mg/kg could be prevented by prior injection with atropine sulfate. It is suggested that both central and peripheral cholinesterase changes are important in determining the nature of the behavioral effects observed after exposure to this compound.     

Investigation of the reported protective effect of cycloheximide on memory

Many findings support the hypothesis that formation of long-term memory requires synthesis of proteins in the nervous system close to the time of learning. This hypothesis has been challenged recently by reports that the protein synthesis inhibitor cycloheximide (CYC) injected 2 hr prior to passive avoidance training in mice or rats attenuated the memory impairment induced by a usually amnestic dose of CYC administered 30 min pretraining. To investigate the reports of a "protective" effect of the prior injection, we attempted to replicate them and test their generality. For replication we administered either paired injections of CYC--120 mg/kg 2 hr prior to training and 30 mg/kg 30 min prior to training--or single injections of CYC (either 120 mg/kg or 30 mg/kg) 30 min pretraining and tested for retention of the passive avoidance habit either 1 or 7 days later. No attenuation of amnesia was observed at 1 day tests. Attenuation of amnesia following the double injection of CYC was observed at 7 day tests. When another protein synthesis inhibitor, anisomycin, was used in the same experimental design, there was no "protective" effect; two injections of anisomycin produced greater memory impairment for the passive avoidance habit than did the single low dose. Also, for active avoidance training, two successive injections of CYC caused significantly greater amnesia than did a single dose; this is the opposite of a "protective" effect. We suggest that the reported "protective" effect of CYC on memory is an as yet unexplained phenomenon that does not generalize to other antibiotic drugs and is specific to the passive avoidance task.     

Effect of lysine vasopressin on pentylenetetrazol-induced retrograde amnesia in rats

Lysine vasopressin (1 microgram/rat SC) administered 1 hr prior to either the acquisition trial or 24 hr retention trial facilitated passive avoidance retention. Amnesia was produced when a single 50 mg/kg (IP) injection of pentylenetetrazol was given immediately following the passive avoidance acquisition trial. A single injection of lysine vasopressin (1 microgram/rat SC) administered 1 hr prior to either the acquisition trial or 24 hr retention trial antagonized the amnesia.     

Pharmacological studies of the new centrally acting muscle relaxant 4'-ethyl-2-methyl-3-pyrrolidinopropiophenone hydrochloride

The pharmacological properties of 4'-ethyl-2-methyl-3-pyrrolidinopropiophenone hydrochloride (HSR-770), a new centrally acting muscle relaxant, were investigated in experimental animals. HSR-770, within a dose range of 1-10 mg/kg i.v., relaxed alpha- and gamma-rigidities in rats, the dose required to relax gamma-rigidity being lower than the dose required to relax alpha-rigidity. When HSR-770 was intraduodenally (i.d.) administered within a dose range of 25-50 mg/kg, the relaxant activity on alpha-rigidity was potent and long-lasting in comparison with eperisone or tolperisone. HSR-770 inhibited flexor reflex more strongly than patellar reflex in a dose-dependent manner within a dose range of 2.5-10 mg/kg i.v. or 25 and 50 mg/kg i.d. in anesthetized cats. In spinal cts, HSR-770 (5 and 10 mg/kg i.v.) inhibited flexor reflex but this potency was weaker than that in anesthetized cats. HSR-770 (12.5-50 mg/kg i.d.) inhibited he crossed extensor reflex in anesthetized rats. In spinal cats, HSR-770 inhibited mono- and polysynaptic reflex potentials to the same extent and also depressed dorsal root reflex potential at 5 and 10 mg/kg i.v. HSR-770 at a dose (50 mg/kg p.o.) which exerted muscle relaxant activity on both rigidity and flexor reflex, had little effect on spontaneous motor activity (mice), hexobarbital-induced sleeping time (mice) and conditioned avoidance response (rats). These results indicate that HSR-770 is a potent centrally acting muscle relaxant and that its central nervous system depressant activity is relatively weak.     

Effects of bifemelane hydrochloride (MCI-2016) on experimental amnesia (passive avoidance failure) in rodents

To further predict the possible activity on memory disorders, the effect of MCI-2016 (bifemelane hydrochloride) was examined using the passive avoidance (PAR) failure technique as an experimental model of amnesia. The amnesia was produced either by post training treatments of electroconvulsive shock (ECS), scopolamine (mice) and cycloheximide or by pre-test injection of scopolamine (rats). In ECS-PAR failure model, the retention test was carried out 3 hr (3 hr experiment) or 24 hr (24 hr experiment) after ECS. MCI-2016 showed a significant improvement when administered just after ECS (3 hr experiment, 30 mg/kg, i.p.) or 0.5 hr before the retention test (24 hr experiment, 10-30 mg/kg, i.p.). Cahopantenate was only active in the 3 hr experiment (500 mg/kg, i.p.), and piracetam was rather active in the 24 hr experiment (60 mg/kg, i.p.). MCI-2016 (30 mg/kg, i.p.) prevented the scopolamine-induced PAR-failure. In this model, physostigmine (0.3 mg/kg, i.p.) exhibited a tendency to improve the failure. In another scopolamine-induced PAR failure model in mice, all of the test drugs showed a significant improvement at different dose levels. The effect of MCI-2016 (25-100 mg/kg, p.o.) was superior to those of piracetam, aniracetam and choline chloride. Higher doses of MCI-2016 were required to improve the cycloheximide-induced PAR failure. Considering the experimental conditions and results, it may be suggested that MCI-2016 ameliorates the amnesia possibly through its influence on memory consolidation and retrieval processes.     

Similar effects of a monoamine oxidase inhibitor and a sympathomimetic amine on memory formation

Amnesia resulting from inhibition of cerebral protein synthesis by cycloheximide can be prevented by subcutaneous injection of the monoamine oxidase inhibitor pargyline (25 mg/kg) or the sympathomimetic amine metaraminol (3.0 mg/kg) administered up to 30 min following learning of a single trial passive avoidance task in day-old chickens. The injection has to be made during the life time of labile memory for the prevention of cycloheximide-induced amnesia. On the other hand, amnesia induced by the Na/K ATP'ase inhibitor ouabain can only be prevented if these two agents are administered up to 5 min after learning, i.e. during the life time of short-term memory. In addition, both agents produce a retrieval deficit 90 min after the injection, but only when memory is in long-term storage. These results are compared to those obtained with administration of norepinephrine, d-amphetamine and diphenylhydantoin.     

Inhibition of cerebral protein synthesis: performance at different times after passive avoidance training.

Inhibition of cerebral protein synthesis impairs long-term memory in a variety of species and tasks. Recently it was reported that subcutaneous injection of the protein synthesis inhibitor cycloheximide impaired short-term retention, measured 10 min after training in a passive avoidance task. To examine the possibility that inhibition of cerebral protein synthesis may sometimes disrupt short-term memory, mice were injected subcutaneously with cycloheximide (120 mg/kg) or anisomycin (150 mg/kg), or bitemporally with cycloheximide or anisomycin (100 mug/side) and given one training trial in a passive avoidance box. Subcutaneously injected cycloheximide reduced step-through latencies 10 min after training as reported previously, but anisomycin or bitemporally injected cycloheximide did not. All 4 drug groups exhibited impaired long-term memory. Since the results obtained at short intervals after training varied depending on the drug and route of injection, the impairment produced by subcutaneous cycloheximide at 10 min after training cannot be attributed to inhibition of cerebral protein synthesis. It is suggested that performance at short intervals after training reflects drug side effects on step-through behavior. By contrast, the impairment obtained at long intervals after training is consistent with the hypothesis that cerebral protein synthesis is required for formation of long-term memory.     

The antihypertensive effect of a selective central muscarinic cholinergic antagonist: N-(4-diethylamino-2-butynyl)-succinimide

N-(4-Diethylamino-2-butynyl)-succinimide (DKJ-21), a cholinergic antagonist selective for muscarinic receptors in the central nervous system, has an antihypertensive effect in conscious spontaneously hypertensive rats (SHR) but not in normotensive Wistar-Kyoto (WKY) control rats. Intravenous doses over a range of 3.1 to 25.0 mg/kg produced a dose-dependent decrease in systolic blood pressure. This effect was still apparent 24 hr after drug administration, but blood pressure returned to predrug levels by 48 hr after injection of DKJ-21. In a dose–response study, the maximum antihypertensive response in a group receiving 25.0 mg/kg was 43 ± 8 mm Hg. Marked variability of the maximum response was observed in all age groups and with all doses. Decreases in blood pressure up to 75 mm Hg were obtained in individual animals. The magnitude of the antihypertensive effect is not age related since 10-, 15-, 22-, and 36-week-old SHR responded to the same degree after injection of 25 mg/kg DKJ-21. Smaller doses (50 μg) of DKJ-21 decreased blood pressure when administered into the lateral cerebral ventricle of SHR. Vascular responses to norepinephrine, acetylcholine, dimethylphenylpiperazinium, angiotensin I, and tyramine were not inhibited by i.v. injection of DKJ-21; however, the centrally mediated pressor response to physostigmine was reduced by 60%.     

Interaction of cycloheximide and diisopropylphosphorofluoridate (DFP) during subchronic administration in rat

Male Sprague-Dawley rats daily treated with DFP (0.5 mg/kg/day, sc) exhibited signs of cholinergic toxicity such as tremors and muscle fasciculations between Days 3 and 5 comparable to those observed 15 min after a single acute signs-producing dose (1.5 mg/kg, sc). Further administration of DFP (0.5 mg/kg/day, sc) for 6-14 days led to tolerance development as evidenced by disappearance of the described toxicity signs. The protein synthesis inhibitor cycloheximide, when given in a nontoxic dose (0.5 mg/kg/day, sc) 1 hr before DFP (0.5 mg/kg/day, sc) administration, potentiated the DFP toxicity and rats died after the fifth injection. DFP-tolerant rats developed toxicity signs when subsequently treated with cycloheximide (0.5 mg/kg/day, sc) and DFP (0.5 mg/kg/day, sc). Each drug when given alone for 4 days caused 30-50% reduction of [14C]valine uptake in vivo into the free amino acids pool as well as its incorporation into proteins of brain and skeletal muscles. A combination of these drugs caused a significantly greater inhibitory effect on [14C]valine incorporation into proteins. Cycloheximide (0.5 mg/kg/day, sc) administered for 4 days did not significantly alter the levels of acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), or carboxylesterase (CarbE) activities but potentiated the DFP-induced inhibition of the activities of these enzymes. It is concluded that the cycloheximide pretreatment potentiates DFP toxicity by a mechanism that is related to inhibition of the synthesis of proteins such as AChE, BuChE, and CarbE.     

Effects of valproic acid derivatives on the acquisition of active avoidance behavior in the rat

The effects of valproic acid derivatives on the acquisition of active avoidance behavior have been studied in the rat. Sodium valproate (50 and 100 mg/kg), magnesium valproate (100 mg/kg) and dipropylacetamide (100 mg/kg) were administered intraperitoneally 1 hr prior to the behavioral test (shuttle-box, single session). Acquisition of active avoidance behavior was facilitated by sodium valproate, unaffected by magnesium valproate, and inhibited by dipropylacetamide. These findings suggest that behavioral effects of valproic acid derivatives cannot be explained only in terms of a general inhibitory action on the central nervous system.     

Effects of subchronic administration of soman on acquisition of avoidance-escape behavior by laboratory rats

Holtzman male Sprague-Dawley rats were given four injections of saline or soman at 31 micrograms/kg or 46 micrograms/kg. The injections were given every 3 days during a 2-week period. Tail vein blood samples, drawn 24 hr before the first injection and 24 hr after the last injection, were analyzed for acetylcholinesterase (AChE) inhibition. For the low and high soman groups, whole blood AChE was inhibited 20 and 24%, respectively, while plasma AChE was inhibited 17 and 25%, respectively. Parallel saline injections produced a small inhibition of whole blood AChE and an increase in activity (negative inhibition) of plasma AChE. During the second week of soman administration rats began training on a discriminated shock avoidance task. The seven animals administered 46 micrograms/kg soman did not learn the lever-pressing avoidance response during a period of 120 days. Five of eight saline rats and four of seven 31 micrograms/kg soman rats learned the avoidance response. There was no significant difference between learners relative to rate of learning of the avoidance task. All of the rats learned to escape the shocks. These data indicate that subchronic soman inhibits learning of new behaviors by laboratory rats.     

Steroid-like anti-inflammatory effect of superoxide dismutase in serotonin-, histamine- and kinin-induced edemata of mice: Existence of vascular permeability regulating protein(S)

Anti-inflammatory drugs (0.25–30 mg/kg), protein synthesis inhibitors or anti-inducer steroid were administered subcutaneously to mice at time 0–6 hr before the initiation of foot edema by chemical mediators. Dexamethasone (1 mg/kg) inhibited serotonin-, histamine-, bradykinin- and kallikreininduced foot edemata 45–60% at 3–6 hr. More than 1 hr of lag time was required for inhibitory effect. SOD (5 mg/kg) began to inhibit the edema as early as 30 min and reached a maximum at 2–3 hr (40–50%) followed by gradual decrease of inhibition. Any of these four models of inflammation was not inhibited by indomethacin (5 mg/kg) through 0–6 hr, proving that these models are insensitive to the inhibitors of prostaglandin synthesis. Cyclohexamide (3 mg/kg), puromycin (8 mg/kg) or actinomycin D (2 mg/kg) reversed the inhibition attained by dexamethasone or SOD. Moreover, the protein synthesis inhibitors and actinomycin D by themselves increase the moderately induced paw swellings of normal mice. Intravenously administered SOD showed the same time course of inhibition as in the subcutaneous experiment by serotonin edema. As the half-life of SOD is only 6 min in the blood stream, this result suggests also the indirect effect of SOD. SOD may not work to release certain anti-inflammatory factor(s) from the granules like lysosomes, because the repeatedly administered SOD was as effective as first injection. Stimulation of adrenals by SOD is exclusive from the rapidity of SOD effect. Progesterone (non-anti-inflammatory steroid which occupies glucocorticoid receptor) diminish the inhibition by dexamethasone, but did not modify the inhibition by SOD. This means that the effect of SOD is not mediated by glucocorticoid receptor. Basing on these observations, the existence of a natural anti-inflammatory protein (X-protein) is supposed. This protein may be perpetually synthesized in vivo to regulate endothelial cell contraction. SOD is possible to prevent the degradation or inactivation of X-protein by active oxygen radicals, and the accumulation of this protein may diminish the vascular permeability. It is possible that X-protein and steroid-induced protein are the same.     

Behavioral effects of NC-1100, 1-(3,4-dimethoxyphenyl)-2-(4-diphenylmethylpiperazinyl) ethanol dihydrochloride--on ambulatory activity, discrete lever-press response and shuttle avoidance response in mice

Single administration of NC-1100 (3, 10 or 30 mg/kg, p.o.) produced no marked change in the ambulatory activity and discrete lever-press avoidance response. However, 100 mg/kg of this drug produced a toxic effect which was characterized by salivation, tremor, clonic convulsion, etc., but none of the mice died. NC-1100 enhanced the ambulation-increasing effect of methamphetamine (2 mg/kg, s.c.) at 3 and 10 mg/kg, while it failed to modify that of scopolamine (0.5 mg/kg, s.c.). NC-1100, at 100 mg/kg, suppressed both the established discrete lever-press and shuttle avoidance responses, probably due to non-specific and toxic effects. When NC-1100 at 10 mg/kg was administered, the response rate increased only in the shuttle avoidance situation in which the avoidance established mice were used. Furthermore, when NC-1100 at 30 mg/kg was administered immediately before the start of the training session, the avoidance rate increased. However, there was no marked change in the avoidance rate in the 2nd training session which was conducted 24 hr after the 1st training session without the drug administration. Additionally, the response rate increased in the 1st and 2nd training sessions after 10 and 30 mg/kg of NC-1100. The present results suggest that NC-1100 may show a behavior-facilitating effect, although its effect is mild and different from that of typical central stimulants such as methamphetamine.     

Behavioral evaluation of psycho-pharmacological and psychotoxic actions of methylxanthines by ambulatory activity and discrete avoidance in mice.

The behavioral effects of methylxanthines, caffeine, theophylline and theobromine, were compared by means of ambulatory activity and discrete lever-press avoidance response in mice. The single oral administration of 10-100 mg/kg of caffeine, 30-300 mg/kg of theophylline, and 10 mg/kg of theobromine significantly increased the mouse's ambulatory activity. However, 1000 mg/kg of theobromine decreased the activity. The ambulation-increasing effect of methamphetamine (2 mg/kg s.c.) was enhanced by coadministration with caffeine (3-100 mg/kg), theophylline (10-300 mg/kg), and theobromine (10-100 mg/kg). On the other hand, comparatively higher doses of caffeine (up to 30 mg/kg) slightly but significantly decreased the avoidance rate without eliciting any significant change in the response rate. Theobromine significantly decreased the response rate at higher than 300 mg/kg, and the avoidance rate at higher than 100 mg/kg. Furthermore, 1000 mg/kg of theobromine was much toxic for mice, i.e., half mice died within a few hr after the end of the 1-hr avoidance session. Theophylline did not produce any significant change in the avoidance response at the dose range of 3-300 mg/kg. These results may indicate the relative order of the centrally stimulant and/or behavioral toxic actions of methylxanthines in human.     

The effects of acute and repeated administration of various antidepressant drugs on clonidine-induced hypoactivity in mice and rats

Small doses of clonidine produce hypoactivity in mice and rats, probably by stimulating pre-synaptic alpha 2-adrenoceptors in the brain. When mice were injected with desmethylimipramine (DMI, 5 mg/kg) before clonidine it attenuated the hypoactivity, while pretreatment with amitriptyline (5 mg/kg) or mianserin (5 mg/kg) potentiated this behaviour. In rats, desmethylimipramine (20 mg/kg) inhibited and mianserin (5 mg/kg) potentiated the clonidine-induced hypoactivity. Amitriptyline (20 mg/kg), however, had no effect on this behaviour in rats. Mice were also given repeated injections of these 3 antidepressant drugs (5 mg/kg twice daily for 14 days) and were tested with clonidine 12 and 60 hr after the final treatment. At 12 hr desmethylimipramine and amitriptyline both attenuated the hypoactivity responses while the reduction by mianserin was marginal (potency DMI greater than amitriptyline greater than mianserin). At 60 hr, however, amitriptyline and mianserin both decreased the clonidine-induced responses while the attenuation by desmethylimipramine was slight (potency amitriptyline congruent to mianserin greater than DMI). In rats, repeated injections of desmethylimipramine (20 mg/kg), administered twice daily for 14 days, attenuated the clonidine-induced hypoactivity 12 hr after the final treatment and this effect persisted for at least 72 hr. Furthermore, the degree of inhibition of the behavioural responses did not correlate with plasma concentrations of desmethylimipramine and persisted after disappearance of the drug from plasma. In conclusion, these data suggest that after repeated injection, desmethylimipramine, amitriptyline and mianserin all produce an adaptive decrease in the function of central alpha 2-adrenoceptors but the time course of this change differs with the individual antidepressant drug administered.     

Propentofylline improves the postischemic stroke index of sensitive gerbils

The new xanthine derivative propentofylline (3-methyl-1-[5′-oxohexyl]-7-propylxanthine), administered intraperitoneally in a dose of 25 mg/kg 10 min before ligation of the left cerebral carotid artery in “sensitive” gerbils, improves the mean stroke index significantly at 4 and 24 hr postadministration. At a dose of 10 mg/kg, propentofylline improves the mean stroke index at all examined times (4, 24, and 72 hr), but at 5 mg/kg, a protective effect was obtained only after 24 hr. The mortality rate was lower in all propentofylline groups than in the control group. These results suggest that propentofylline is worth further study as a possible antistroke agent.     

Role of cholinergic and GABAergic neuronal systems in cycloheximide-induced amnesia in mice

The role of cholinergic and GABAergic neuronal systems on the cycloheximide (CXM)-induced amnesia was investigated using the step-down-type passive avoidance task in mice. CXM (7.5-120 mg/kg, SC) given just after the training caused amnesia (indicated by short latency to step down from the platform on the grid floor) in the retention test conducted 24 hr later in a dose-dependent fashion. In the CXM (60 mg/kg)-treated mice, a choline esterase inhibitor, physostigmine (PHY; 0.125 and 0.25 mg/kg, IP), or GABA agonists, muscimol (1 and 2 mg/kg, IP) and baclofen (6 and 12 mg/kg, IP), given just after training markedly prolonged step down latency (SDL), indicating reversal of amnesia. The antiamnesic action of PHY (0.125 mg/kg) was almost completely antagonized by a central acetylcholine antagonist, scopolamine (3 mg/kg, SC), but not by a peripheral acetylcholine antagonist, butylscopolamine (3 mg/kg, SC). Furthermore, the antiamnesic action of muscimol (2 mg/kg) was reversed by GABA antagonists, picrotoxin (0.5 mg/kg, SC) and bicuculline (0.5 mg/kg, SC), while the effect of baclofen (12 mg/kg) was reversed by picrotoxin (0.5 mg/kg), but not by bicuculline (0.5 mg/kg). These results suggest that the dysfunction of cholinergic and GABAergic neuronal systems play an important role in the CXM-induced memory impairment on the passive avoidance task.     

Inhibitory influence of dihydroergosine on the aggressiveness of rats and mice

Fifty mg/kg of ergot alkaloid dihydroergosine (DHESN) inhibited the mouse-killing behavior of isolated male rats, while 10 mg/kg did not. This effect of DHESN (50 mg/kg) lasted for 24 hr. When an additional injection of DHESN (50 mg/kg) was given to mouse-killer rats 6 days following the first, the mouse-killing behavior was again inhibited. The effect of the second drug injection also persisted for 24 hr and was accompanied by an increased concentration of 5-HT in the raphe nuclei and hypothalamus and by a decreased concentration of GABA in the olfactory bulbs. DHESN also inhibited aggressiveness in isolated mice. Two hr following the administration of 10 mg/kg DHESN the fighting was inhibited in 46% of pairs tested, while 50 mg/kg abolished it completely. The effect of 50 mg/kg lasted 24 hr. These results, showing the antiaggressive effects of DHESN, support our previous suggestion that DHESN might presumably be a new antidepressant, and suggest that besides the serotoninergic, the GABA-ergic system might also be involved in the modification of behavior induced by this drug.     

Loss of murine tumor thymidine kinase activity in vivo following 5-fluorouracil (FUra) treatment by incorporation of FUra into RNA.

The effects of 5-fluorouracil (FUra) treatment on thymidine kinase (TKase) activity were examined in vivo in CD8F1 mice bearing first generation CD8F1 mouse mammary tumors. TKase activity was not affected by low dose FUra25 (25 mg/kg), a dose which substantially inhibited thymidylate synthase (TSase), but was severely inhibited 24 hr following treatment with FUra100, a weekly maximally tolerated dose, as judged by activity measurements and labeling of DNA with [3H]thymidine. The amount of (FU)RNA was increased markedly with increasing FUra dose from 0.4 nmol/mg DNA at FUra25 to 2.2 nmol/mg DNA at FUra100. At FUra100, TKase activity gradually declined over 24 hr to less than 10% of the control value, remained low for a further 48 hr, and then was gradually restored to control levels by 168 hr. The loss of TKase activity followed the incorporation of FUra into RNA which peaked at 4-5 hr. TKase activity was not restored by removal of endogenous inhibitors but was restored by treatment with uridine. TKase activity was not inhibited by therapeutic levels of methotrexate (300 mg/kg). TKase from murine colon 38 carcinoma was also severely inhibited, but the activity from colon 26 was only partially (50%) inhibited. Ornithine decarboxylase was also inhibited by FUra100 treatment in the CD8F1 tumor. These results demonstrate that certain short-lived, proliferation-related enzymes are affected by FUra doses higher than those required for TSase inhibition, and this effect appears to correlate with incorporation of FUra into RNA. Thus, in some tumors high doses of FUra can inhibit salvage as well as de novo synthesis of thymidylate providing an increased block of DNA synthesis and increased therapeutic advantage.