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At least 19 recordsLinked to original sources
PubMed1989

Cycloleucine blocks NMDA responses in cultured hippocampal neurones under voltage clamp: antagonism at the strychnine-insensitive glycine receptor.

1. Radioligand binding studies have demonstrated that the neutral amino acid cycloleucine may act as a competitive antagonist at the glycine modulatory site on the N-methyl-D-aspartate (NMDA) receptor complex. In the present study, we examined the effects of cycloleucine on NMDA-evoked inward current responses in dissociated hippocampal neuronal cultures using the whole cell voltage-clamp technique. 2. In the presence of 1 microM glycine, cycloleucine caused a reversible, dose-dependent inhibition of NMDA responses with an IC50 of 24 microM. An increase in glycine to 100 microM resulted in a shift to the right of the cycloleucine concentration-effect curve (IC50, 1.4 mM). However, with cycloleucine concentrations less than or equal to 100 microM, a fraction of the block could not be overcome by glycine even at concentrations as high as 1 mM. 3. The cycloleucine block was unaffected by shifts in the holding potential (-60 to +60 mV), and there was no effect of cycloleucine on the reversal potential of the NMDA-evoked current. 4. Cycloleucine failed to effect kainic acid- and quisqualic acid-evoked currents at concentrations which inhibited NMDA responses. 5. We conclude that cycloleucine is a potent and selective antagonist of NMDA-receptor mediated responses. Although this effect occurs in part via competitive antagonism at the glycine modulatory site, the cycloleucine block cannot be completely reversed by glycine indicating an interaction with an additional site on the receptor-channel complex.

Amino Acids↗
PubMed1983

Particles of reduced infectivity and deficient in envelope glycoproteins are produced in cycloleucine-treated B77 avian sarcoma virus-infected chicken embryo fibroblasts.

We have previously shown that the inhibition of methylation reactions by the treatment of B77 avian sarcoma virus-infected cells with medium containing cycloleucine results in an inhibition in the intracellular accumulation of the spliced subgenomic mRNA for the virion envelope protein precursor, whereas the genome-size RNA accumulates in larger than normal amounts (C. M. Stoltzfus and R. W. Dane, J. Virol. 42:918-931, 1982). To measure the production of virus particles, we have now determined the reverse transcriptase activity in the culture fluid from infected cells treated with various concentrations of cycloleucine. The activity was somewhat greater in the fluid from the cycloleucine-treated cells than it was in the fluid from the control cells, suggesting an enhancement of particle production in the presence of cycloleucine. In contrast, the production of infectious virions, as determined by the focus assay, decreased when the cycloleucine concentration of the medium increased. We determined the polypeptide compositions of purified particles produced from infected cells treated with or without cycloleucine and labeled with [(3)H]leucine. The relative amounts of radioactivity associated with p19 and p27 were approximately the same in all of the preparations. In contrast, significant decreases were observed in the relative amounts of [(3)H]leucine radioactivity associated with the virion glycoproteins gp85 and gp37. The extent of the decrease in the ratio of gp85 to p27 was a function of the cycloleucine concentration and correlated well with the decrease in the infectivity of the virus particles. Therefore, it is probable that the observed reduction of specific infectivity results from the reduced amounts of envelope glycoproteins in the particles budding from cycloleucine-treated cells.

Amino Acids↗
PubMed1983

Pharmacological, conformational and dynamic properties of cycloleucine-2 analogues of oxytocin and [1-penicillamine]oxytocin.

The solid phase syntheses of [2-cycloleucine] oxytocin and [1-penicillamine, 2-cycloleucine] oxytocin are reported. [1-Penicillamine, 2-cycloleucine] oxytocin is an oxytocin antagonist exhibiting no in vitro oxytocic activity. In the in vitro oxytocic assay, [1-penicillamine, 2-cycloleucine] oxytocin has a pA2 value of 6.70 +/- 0.08. [2-Cycloleucine]-oxytocin is a full oxytocin agonist exhibiting 4.9 +/- 0.5 U/mg of oxytocic activity. Neither compound possesses any measurable agonist or antagonist activity in the rat pressor assay. Carbon-13 nuclear magnetic resonance chemical shift parameters and spin-lattice relaxation times (T1) of the antagonist, [1-penicillamine, 2-cycloleucine] oxytocin, indicate that the antagonist exhibits similar conformational and dynamic properties as other oxytocin inhibitors previously studied. The carbon-13 nuclear magnetic resonance shift parameters and spin-lattice relaxation times (T1) of the oxytocin agonist, [2-cycloleucine] oxytocin, indicate that the agonist exhibits similar conformational and dynamic properties as oxytocin. These results are discussed in terms of the different receptor requirements for agonist and antagonist activities. It appears that there are different structural and conformational requirements at the 2-position for oxytocic agonist and antagonist activities.

Animals↗
PubMed1978

Experimental cystinuria: the cycloleucine model. II. Amino acid efflux from intestinal and renal tissues.

Loading and unloading experiments using intestinal sacs and renal cortex slices were undertaken to ascertain the role of amino acid efflux in cycloleucine-induced amino-aciduria. The presence of cycloleucine, lysine, or valine on the luminal or antiluminal side of the intestine caused an increased leakage of [14C] cycloleucine, [14C] lysine, and [35S] cystine from the tissue. Similar results were obtained when using kidney cortex slices, except for cystine efflux. The latter phenomenon was inhibited by cycloleucine and lysine. Data, also obtained with renal cortex slices, suggest that cystine and cysteine are recognized by different transport sites although one (the oxidized form) may be typically extracellular and the other (the reduced form), intracellular. A comparison of these data with previous works done in our laboratory shows that cycloleucine affects efflux less than influx and further suggests that in rats given cycloleucine, renal transport is impaired only at the brush border level for cystine and at both luminal and antiluminal membranes for dibasic amino acids.

Amino Acids↗
PubMed1984

Evidence that cycloleucine affects the high-affinity systems of amino acid uptake in cultured human fibroblasts.

The influence of cycloleucine on kinetic parameters of uptake of L-alanine, L-proline and L-leucine into cultured human fibroblasts was examined under initial-rate conditions with substrate concentrations of 0.05-10 mM and 5 mM-cycloleucine. Kinetic data obtained by computer analysis showed that, in the absence of cycloleucine, cell uptake was heterogeneous for each amino acid. L-Alanine and L-leucine entered by two transport systems with different affinities; L-proline was taken up by one saturable transport system plus a diffusion-like process. This heterogeneity disappeared in the presence of cycloleucine, since the high-affinity systems were no longer detectable. The remaining process had the same kinetic constants as the low-affinity system for alanine and leucine and a KD similar to the diffusion constant for proline. The influence of cycloleucine on the amino acid uptake was not specific either to the amino acid concerned or to a particular transport system, since the three neutral amino acid-transport systems, A, ASC and L, were involved in these experiments. This influence was shown to be unaffected by the absence of Na+ (for leucine uptake). ATP content of the cells was identical in the presence or in the absence of cycloleucine.

Adenosine Triphosphate↗
PubMedDate not supplied

Comparison of doxorubicin with cycloleucine in the treatment of sarcomas.

In this patient series, doxorubicin and cycloleucine at a dose of 300 mg/kg both show response rates in the treatment of advanced soft tissue sarcomas of about 15%. Lower doses of cycloleucine (200 mg/kg) yielded less toxicity but were less effective against the sarcomas (6% response rate, three of 51 patients). There were no complete responses with cycloleucine and there were three with doxorubicin. Survival times for patients receiving doxorubicin were significantly longer than those of patients receiving cycloleucine at doses of 300 mg/kg (P less than 0.001) or 200 mg/kg (P = 0.02). The estimated survival times were 29 weeks for doxorubicin and 21 (300 mg/kg) and 18 (200 mg/kg) weeks for cycloleucine. Toxic effects due to cycloleucine were excessive, with severe thrombocytopenia and central nervous system depression being the most prominent.

Adult↗
PubMed1975

Cycloleucine (1-amino-cyclopentane carboxylic acid): tubular reabsorption and inhibitory effect on amino acid transport in the rat kidney. (Microperfusion experiments).

Renal tubular reabsorption of cycloleucine (1-amino-cyclopentane carboxylic acid) was studied in vivo et situ by continuous microperfusion of single proximal tubules of the rat. The results show: a) cycloleucine is reabsorbed rapidly compared with other amino acids b) this reabsorption is saturable and can be inhibited by oligomycin c) cycloleucine inhibits tubular reabsorption of L-arginine, glycine, and of L-phenylalanine. Mutual reciprocal inhibition occurs only with L-phenylalanine (and perhaps also with glycine). A maximal possible permeability coefficient for cycloleucine (less than 6 times 10--5 cm times sec--1) was calculated. Assuming simple 2-parameter kinetics, Vmax and Km for tubular reabsorption of cycloleucine were estimated. It can be concluded from the present results that cycloleucine is reabsorbed by a mechanism that transports L-phenylalanine, but not by the system shared by dibasic amino acids.

Absorption↗
PubMed1975

Amino acid accumulation in frog muscle. II. Are cycloleucine fluxes consistent with an adsorption model for concentrative uptake of amino acid?

Cycloleucine accumulation by frog muscle was studied at 0 degrees C and 25 degrees C. At external concentrations less than 5 mM the distribution ratio of cycloleucine is higher at 0 degrees C. At concentrations greater than 5 mM the converse is true due to apparent exclusion of cycloleucine from a larger portion of the cell water at 0 degrees C than at 25 degrees C. The steady state data are consistent with an adsorption model for amino acid accumulation. Flux studies provide a means to rule out this model if all the possible rate-limiting steps in the movement of amino acid into and out of the cell are considered. These steps include intra-cytoplasmic diffusion, desorption from cytoplasmic or membrane sites and passage through the cell membrane. The assumption is made that the rate-limiting step for influx and efflux is the same, allowing the use of either influx or efflux data to examine the model. Diffusion-limited flux is ruled out on the basis of "influx profile analysis" of the time course of cycloleucine entry at both 0 degrees C and 25 degrees C. At least 95% of all intracellular cycloleucine leaves frog muscle cells with a single exponential time course at both 0 degrees C and 25 degrees C. The rate constant of efflux does not vary with cellular concentration. These findings are shown to be incompatible with desorption-limited efflux. They are compatible with membrane-limited efflux only if (i) adsorption sites are located on membranes with direct access to the extracellular space and (ii) the rate constant for desorption is equal to the rate constant of membrane-limited efflux of free amino acid. It is considered unlikely that such a coincidence would occur at both 0 degrees C and 25 degrees C. Therefore, an adsorption model for cycloleucine accumulation in frog muscle appears to be untenable.

Amino Acids↗
PubMed1978

Methionine metabolism in BHK cells: preliminary characterization of the physiological effects of cycloleucine, an inhibitor of s-adenosylmethionine biosynthesis.

Cycloleucine is in vitro a competitive inhibitor of methionine adenosyltransferase, an enzyme involved in S-adenosylmethionine biosynthesis. The physiological effects of this drug on baby hamster kidney cells have been studied. When cells are grown in a medium containing 10 micron methionine, cycloleucine is an inhibitor of cell proliferation; high concentrations of methionine are able to withdraw this inhibition suggesting that cycloleucine toxicity is related to methionine metabolism. The drug does not primarily affect methionine uptake and its subsequent use for protein biosynthesis. Cycloleucine toxicity is correlated with a block of SAM biosynthesis and nucleic acids methylations. The actions of cycloleucine on progression in the cell cycle and DNA, RNA and protein biosynthesis are studied. The implications of these results are discussed.

Amino Acids↗
PubMed1978

Selection and preliminary characterization of cycloleucine-resistant CHO cells affected in methionine metabolism.

Cycloleucine is in vivo a potent inhibitor of S-adenosylmethionine (SAM) biosynthesis and subsequent methylation reactions in somatic mammalian cells. Cycloleucine-resistant (CLr) clones were isolated from CHO cells by single-step selection. Their phenotype was stable when they were grown in the absence of drug. These clones appeared randomly in cultures at the frequency of 5 x 10(-6)/cell/generation, as determined by a fluctuation test. EMS mutagenesis did not significantly increase this frequency. The cycloleucine-resistant phenotype was codominant in intraspecific hybrids. Cycloleucine-resistant clones showed increased SAM pools; on the contrary, methionine pools were not significantly affected in these clones when compared to the wild-type cells. This increased SAM production was correlated with an increase of methionine adenosyltransferase (MAT) SPECIFIC ACTIVITY IN RESISTANT CLONES UNDER VARIOUS GROWm. The mechanism of posttranscriptional control of MAT biosynthesis was not affected in the cycloleucine-resistant clones nor were the kinetic properties of the enzyme modified. The genetic or epigenetic origin of this resistance mechanism is discussed.

Amino Acids↗
PubMed1991

Cadmium inhibition of basolateral solute fluxes in rabbit renal tubules and the nature of cycloleucine uptake.

The objective of this investigation was to determine whether Cd inhibition of amino acid transport across basolateral (BL) cell membranes in renal tubules results from a direct toxic action at that site. The concentration ratio (R) of cycloleucine in cell water/arterial plasma at steady state in nonfiltering rabbit kidneys consistently exceeded 1.0, thus confirming the active nature of BL amino acid uptake. BL cycloleucine extrusion, though down the concentration gradient, has previously been shown to be greatly slowed in Cd-poisoned animals; nevertheless, R remained unchanged. Active uptake and passive extrusion of cycloleucine must therefore be equally sensitive to Cd, a fact strongly suggesting an indirect action of the metal on BL solute transfer. This hypothesis is strengthened by the observation that R for paraaminohippurate (PAH), another solute actively accumulated across BL membranes, also remained unaffected by Cd poisoning. The reduction in R(PAH) by the direct transport inhibitor probenecid served as positive control. The additional finding that R(cycloleucine) is also depressed by probenecid, as well as by excess PAH, indicates some overlap in substrate specificities of the two carrier systems. The systems are not identical, however, as can be deduced from the observation that L-leucine affected only transport of cycloleucine, not that of PAH.

Animals↗
PubMed1986

Echinococcus granulosus: absorption of cycloleucine and alpha-aminoisobutyric acid by protoscoleces.

Protoscoleces of Echinococcus granulosus absorb the amino acids cycloleucine and alpha-aminoisobutyric acid (AIB) by a combination of mediated uptake and diffusion. After correcting for the latter, values for Kt and Vmax of 0.124 mM and 0.947 nmoles/mg protein/2 min for cycloleucine were calculated; corresponding values for AIB were 0.039 mM and 0.139 nmoles/mg protein/2 min. Both amino acids were accumulated against a concentration gradient and a comparison of Kt and Ki values determined in mutual inhibition experiments suggested that both cycloleucine and AIB share a common uptake locus (loci). Cycloleucine uptake was pH-dependent and could be inhibited by a variety of other amino acids. Neither D- nor L-proline inhibited cycloleucine absorption but D-methionine, D-alanine, D-leucine, D-valine and D-serine were much more effective inhibitors than their L-counterparts.

Absorption↗
PubMed2000

Cycloleucine fluxes during rat vasa recta and loop microinfusions in vivo and loop microperfusions in vitro.

Amino acids are apparently recycled between loops of Henle and vasa recta in rat papilla in vivo. To examine this process in the absence of metabolism, we performed continuous microinfusions of rat renal papillary ascending thin limbs (ATLs) and vasa recta in vivo, and microperflusions of isolated rat renal papillary descending thin limbs (DTLs) and ATLs in vitro using the nonmetabolizable, synthetic, neutral amino acid cycloleucine. Like naturally occurring amino acids, approximately = 25% of radiolabeled cycloleucine microinfused into ATLs in vivo was reabsorbed by a process that was not saturable or inhibitable. Also, like naturally occurring amino acids, approximately = 47% (relative to inulin) of radiolabeled cycloleucine microinfused into ascending vasa recta in vivo was transferred directly into ipsilateral tubular structures (probably DTLs) by a saturable and inhibitable process. In DTLs perfused in vitro, unidirectional bath-to-lumen fluxes (Jbl) tended to exceed unidirectional lumen-to-bath fluxes (Jlb), whereas in ATLs perfused in vitro Jlb tended to exceed Jbl, but the differences were not statistically significant. Moreover, none of the unidirectional fluxes was saturable or inhibitable, an observation compatible with apparent reabsorption from ATLs in vivo but incompatible with apparent movement from vasa recta to DTLs in vivo. These in vitro observations are like those made previously for the naturally occurring neutral amino acid L-alanine. The lack of saturation and inhibition, like the previous data on L-alanine, suggest that transepithelial movement of amino acids in thin limbs of Henle's loop may occur via a paracellular route and that regulation of amino acid movement in vivo may involve vasa recta, not DTLs. They also suggest that cycloleucine is a good nonmetabolizable surrogate for the study of neutral amino acid transport in the kidney.

Alanine↗
PubMed1978

Effect of cycloleucine on amino acid accumulation by human diploid fibroblasts.

Cycloleucine is a synthetic amino acid which produces, in vivo, biochemical abnoramlities comparable to those seen in human cystinuria-lysinuria. The effect of cycloleucine on intracellular accumulation of amino acids overlapping separate transport systems was studied using human diploid fibroblasts subcultures on glass coverslips. The data indicated that alpha-alanine, serine and proline accumulation was inhibited significantly by cycloleucine. The percentage of inhibition was approximately the same. Lysine was less affected by cycloleucine, but this amino acid accumulation proceeded at a rate slower than for neutral amino acids. In vitro, this inhibitory effect seems to be a generalized phenomenon affecting substrates. These results confirm in human fibroblasts data reported for human and rat kidney slices.

Amino Acids↗
PubMed1992

Uptake of alpha-aminoisobutyric acid and cycloleucine on skeletal muscles in burned rats.

The uptake of alpha-aminoisobutyric acid (AIB) and aminocyclopentane-carboxylic acid (cycloleucine) was studied in soleus muscles isolated from small rats (body weight 50-60 g) during the first 6 h after a major thermal injury, the so-called 'ebb phase' or 'hypometabolic phase'. Soleus muscles were dissected intact from rats at 0.5, 1, 3 and 6 h after extensive deep burn injury (30 per cent TBSA) and then incubated for 2 h in Krebs-Henseleit bicarbonate buffer (pH 7.4), 5.5 mM glucose, bovine serum albumin and radiolabelled AIB or cycloleucine. The results were expressed as the distribution ratio of AIB or cycloleucine between intracellular and extracellular fluid. The AIB uptake in vitro was found to be significantly increased (30 per cent) in the initial half hour postburn only, and then slowly reduced during the subsequent hours to near the value found in non-burn animals. Muscle cycloleucine uptake in vitro showed no significant change in these studies. In our second study, extensor digitorum longus leg muscle were incubated in Krebs-Henseleit bicarbonate buffer with different pH values (7.2-7.5). No significant difference was found in muscle AIB uptake. In summary, since muscle amino acid uptake remained relatively stable during this period, it is suggested that the alteration of amino acid transport across muscle cells may not be a contributing factor to the alteration of amino acid flux during the early phase of stress.

Aminoisobutyric Acids↗
PubMed1975

Cycloleucine transport in isolated rat thymocytes: in vitro effects of triiodothyronine and thyroxine.

Thymocytes obtained from suckling or young adult rats were used as a model system to study the action of thyroid hormones in vitro. In this tissue, L-triiodothyronine (T3) increased the uptake of the non-metabolizable amino acids, alpha-aminoisobutyrate and cycloleucine. A detectable effect of T3 on the uptake of cycloleucine was seen at a concentration of 0.1 muM and maximum effects were seen at 20 muM. Thyroxine (T4) also increased cycloleucine uptake with about one-third the potency of T3, and this effect could not be ascribed to conversion of T4 to T3. In contrast, L-monoidotyrosine and L-diiodotyrosine were without effects on transport. Kinetic studies indicated that T3 enhanced uptake by inhibiting amino acid efflux; no effect was seen on influx. The effect of T3 on amino acid uptake was evident within 1 min, and was not inhibited by either prior treatment of the cells with cycloheximide or by lowering the incubation temperature from 37 to 24 C. In other studies, when T3 was injected into rats in vivo at a dose of 20 mug/100 g, the uptake of cycloleucine was enhanced in thymocytes obtained 1 h later. These data suggest that thyroid hormones can directly influence amino acid transport in rat thymocytes. This effect is prompt, is independent of new protein synthesis, and may reflect a direct interaction with specific components of the cell membrane.

Animals↗
PubMed1990

Effects of conformational constraint in 2- and 8-cycloleucine analogues of oxytocin and [1-penicillamine] oxytocin examined by circular dichroism and bioassay.

The analogues of oxytocin and [1-penicillamine]oxytocin, containing a cycloleucine (Cle) residue in position 2 or 8, were investigated by means of circular dichroism measurements in different solvents, and the results examined in terms of their biological activities. A cycloleucine residue in position 2 substantially reduces the free conformational space of the hormone 20-membered ring moiety (including the disulfide group), and stabilizes a conformation which is close to one of the possible conformations of oxytocin and involves a beta-turn. In position 8, the Cle residue affects the conformation of the Tyr2 side chain, apparently forcing it away from the space above the 20-membered disulfide ring. However, it does not appear that the Cle residue has any significant effect on the overall backbone conformation of the hormone. The steric effect of the penicillamine residue in position 1 on the conformation of the disulfide group and Tyr2 side chain from previous investigations is further confirmed. The synthesis and biological potency of [1-penicillamine, 8-cycloleucine]oxytocin is described. This analogue exhibits a strong inhibitory effect on the uterotonic activity of oxytocin in vitro. It also inhibited the vasopressor response to vasopressin.

Animals↗
PubMed1978

Cycloleucine blocks 5'-terminal and internal methylations of avian sarcoma virus genome RNA.

Cycloleucine, a competitive inhibitor of ATP: L-methionine S-adenosyltransferase in vitro, has been used to reduce intracellular concentrations of S-adenosylmethionine and by this means to inhibit virion RNA methylation in chicken embryo cells that are infected with B77 avian sarcoma virus. Under conditions of cycloleucine treatment, where virus production as measured by incorporation of radioactive precursors or by number of infectious particles is not significantly affected, the internal m6A methylations of the avian sarcoma virus genome RNA are inhibited greater than 90%. The predominant 5'-terminal structure in viral RNA produced by treated cells in m7G(5')pppG (cap zero) rather than m7G-(5')pppGm (cap 1). It appears from these results that internal m6A and penultimate ribose methylations are not required for avian sarcoma RNA synthesis and function. Furthermore, these methylations are apparently not required for transport of genome RNA to virus assembly sites. The insensitivity of the 5'-terminal m7G methylation to inhibition by cycloleucine suggests that the affinity of S-adenosylmethionine for 7-methylguanosine methyltransferase is significantly greater than for the 2'-0-methyltransferases or the N6-methyltransferases.

Amino Acids↗