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At least 19 recordsLinked to original sources

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↗

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↗

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↗

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↗

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↗

PubMed1978

Biosynthesis and utilization of extensively undermethylated poly(A)+ RNA in CHO cells during a cycloleucine treatment.

The role of RNA methylations in the control of mRNA maturation and incorporation into polysomes has been investigated through a study of the effects in vivo of cycloleucine, a specific inhibitor of S-adenosyl-methionine mediated methylation. During the cycloleucine treatment, the rate of biosynthesis of hnRNA and its subsequent polyadenylation were only slightly reduced as compared with untreated cells. However a significant lag-time in the cytoplasmic appearance of poly(A)+ undermethylated molecules was observed, in parallel with a transient shift in the average size of hnRNA towards higher molecular weight. Nevertheless, the total amount of pulse-labelled poly(A)+ mRNA transferred to cytoplasm after a long chase time (3 h.) was approximately the same for both cycloleucine-treated and control cells. Extensively undermethylated poly(A)+ cytoplasmic RNAs, possessing a 5' terminal cap were incorporated into polysomes in proportions very similar to control messenger molecules. These results suggest that a normal level of methylation is not stringently required for the production of the functional mRNA molecules although it appears to be of importance for the kinetics of the maturational process.

Amino Acids↗

PubMed1975

Experimental cystinuria: the cycloleucine model. I. Amino acid interactions in renal and intestinal epithelia.

The injection of cycloleucine (1-aminocyclopentanecarboxylic acid (ACPC) into rats produces a hyperexcretion of dibasic amino acids and cystine, an aberration resembling cystinuria. This may constitute a model of experimental cystinuria, and the transport of amino acids involved in this disease was studied with the techniques of everted intestinal sacs (in vitro) and microinjections into renal tubules (in vivo). In verted sacs from normal rats, there was a decrease in transfer and in accumulation of L-cystine (0.03 mM) and L-valine (0.065 mM) when ACPC was on the mucosal (luminal) side. Dibasic amino acids such as L-arginine and L-lysine caused a similar inhibition of the transport of L-cystine. However, when ACPC was on the serosal (antiluminal) side, a lesser effect was noted while arginine and lysine had no effect. Intestinal sacs from treated rats (ACPC, 300 mg/kg X 3 days) transferred and accumulated as much L-cystine as those from control rats. The interaction between cycloleucine and L-cystine was competitive at the luminal and non-competitive at the antiluminal side of the intestine. Cycloleucine inhibited L-lysine transport in a non-competitive fashion at either side of the intestine. L-Lysine also interacted in a non-competitive fashion with L-cystine transport at the luminal membrane. In proximal convoluted tubules, the presence of L-arginine or ACPC caused a decrease in the transport of L-cystine and L-lysine. L-Valine exerted no effect. Furthermore, L-lysine and ACPC did not impair the reabsorption of L-valine significantly. These results suggest a functional heterogeneity between luminal and antiluminal membranes of renal and intestinal epitehlia and the existence, at both membranes, of different transport sites for cystine and dibasic amino acids.

Amino Acids↗

PubMed1979

Pathways of cycloleucine transport in killifish small intestine.

By using blocking concentrations of competitors, i.e., concentrations of other neutral amino acids that cause maximal inhibition, cycloleucine transport into slices or everted sacs of killifish (fundulus heteroclitus) small intestine could be partitioned into three pathways. One is apparently not mediated, a second is inhibited by all neutral amino acids tested (component 1), and a third, is inhibited by alpha-aminocarboxylic acids (component 2), but not by beta-alanine or taurine. Both mediated pathways were Na dependent, and each yielded a linear double reciprocal plot of initial slice uptake vs. cycloleucine concentration. Apparent Kt and Vmax values for component 1 were 0.03 mM and 33 pmol/mg tissue per 3 min, respectively; corresponding values for component 2 were 0.12 mM and 28 pmol/mg tissue per 3 min. Additional experiments with an intestinal brush border membrane vesicle preparation indicate that these mediated components reflect true differences in carrier specificity rather than the differential effects of inhibitors on metabolism or on the Na gradient that drives cycloleucine transport.

Amino Acids↗

PubMed1976

[Effect of cycloleucine on renal uptake of dibasic amino acids and cystine].

Cycloleucinead ministration (a synthetic amino acid) to rats produces a selective hyperaminoaciduria bearing on dibasic amino acids (lysine, arginine, ornithine) and cystine. Interference of cycloleucine with tubular reabsorption of these amino acids was studied "in vitro", using rat kidney cortex slices. When dibasic amino acids and cystine are at physiological concentrations, the data indicate that cycloleucine decreases their intracellular accumulation. However, effect of cycloleucine "in vitro" is not specific whatsoever its concentration; similar results are obtained with neutral amino acids such as alpha-alanine and valine. Inhibitory effects are not modified by the presence of several amino acids in the incubation medium. The results are interpreted as evidence for a dissociation between "in vitro" cellular accumu-lation and "in vivo" transepithelial transport.

Amino Acids↗

PubMed1975

Microperfusion study of the kinetics of reabsorption of cycloleucine in early and late segments of the proximal convolution of the rat nephron.

The proximal tubular reabsorptive capacity for the non-metabolizable amino acid, cycloleucine, was studied in the rat nephron by stationary microperfusion. Tubular reabsorptive rates were greatest near the glomerulus and declined progressively along the convolution. A kinetic analysis of cycloleucine reabsorption in terms of luminal concentration revealed that this reduced transport rate was associated with an increase in the half-saturation constant of the kinetic curve, rather than a decrease in the maximum transport capacity. Since our previous findings with the metabolizable amino acid, -L-histidine, were identical we can conclude that this decline in reabsorption of neutral amino acids as a function of distance along the convolution is an intrinsic property of the transport system and is not related to tubule cell amino acid metabolism. The transport curves for cycloleucine absorption did not give a simple Michaelis-Menten relation but rather followed a course suggesting that more than one transport system might be involved.

Absorption↗

PubMed1976

Cycloleucine uptake in the brainstem of thiamine-deficient rats.

Immature female rats were subjected to acute dietary deficiency of thiamine. An autoradiographic method was used in the semi-quantitative determination of concentration of 1-aminocyclopentane-1-carboxylic acid-carboxylic-14C (cycloleucine) in brainstem regions after intravenous administration of tracer quantities. The time course of tissue concentrations was followed and compared with that of normal and isocaloric control animals. Our data indicate that thiamine deficiency of sufficient magnitude to induce brainstem lesions has an effect on the transport of cycloleucine. The initial 2 min values in the nuclear areas are appreciably reduced whereas the 6 min values are significantly elevated as compared with controls; no change was demonstrated in the white matter of the inferior cerebellar peduncle. The effect on transport appears to be more on the mechanism of efflux than of influx in terms of blood brain barrier function.

Amino Acids↗

PubMedDate not supplied

Enhancement by glucocorticoid deficiency of the increase in cycloleucine accumulation induced in rat thymocytes by triiodothyronine.

In previous studies, we have demonstrated that 3,5,3'-triiodothyronine (T3), either added to suspended media in vitro or injected acutely in vivo, increases the in vitro accumulation of the non-metabolized amino acid cycloleucine (CLE) by thymocytes harvested from weanling rats. We now report that this reponse is greatly enhanced by prior adrenalectomy of the donor rat. In vitro, a significant increase in CLE accumulation in thymocytes from adrenalectomized rats was induced by T3 at a concentration of 1 x 10-10 M, while a concentration of 1 x 10-6 M was required to produce a similar and significant effect in thymocytes from intact animals. In adrenalectomized animals, a single iv dose of T3 (0.5 microgram/100 G bw) significantly increased the in vitro accumulation of CLE in thymocytes harvested two hours later. In contrast, ten-times that dose was ineffective in control animals. Increased sensitivity to T3 was abolished by physiological replacement doses of hydrocortisone. The data are consistent with the well-known opposing effects of physiological levels of thyroid and glucocorticoid hormones on the growth and function of lymphoid tissue in vivo and, together with other findings, suggest that thyroid hormones modulate the cellular accumulation of amino acids in the intact animal.

Adrenalectomy↗

PubMed1977

RNA methylation and control of eukaryotic RNA biosynthesis. Effects of cycloleucine, a specific inhibitor of methylation, on ribosomal RNA maturation.

The role of RNA methylation in the control of ribosome production in mammalian cells has been reinvestigated through a study of the effects in vivo of cycloleucine, a specific and reversible inhibitor of nucleic acid methylations. No close coupling is observed between methylation and transcription. During an extensive blocking of methylation, the synthesis of preribosomal 45-S RNA continues, although at a slightly reduced rate. Transcription and methylation can be temporarily uncoupled in vivo without impairing significantly the efficiency of the subsequent maturation of the transcript which takes place when the methylation is resumed. At the post-transcriptional level, two main observations are made. First, the cleavage pattern of ribosomal RNA is not qualitatively modified by the drug treatment. Preribosomal 45-S RNA which is synthesized during an extensive blocking of methylations (95%) is cleaved in vivo in a stepwise fashion into molecules resembling the species of the normal processing. Undermethylation does not result in the appearance of new size products and no extensive or rapid degredation of rRNA precursors can be detected. Second, the global efficiency of the process of ribosomal RNA maturation is severely affected, in quantitative terms; the undermethylation partially inhibits several stages in the maturation pathway and does not block selectively at a definite step of processing, as previously reported by others. Two main modifications are observed. (a) In the nucleus, the life times of the various undermethylated intermediates of ribosomal RNA maturation are markedly increased and a significant accumulation of these forms is detected by long-term labelling studies. (b) The rate of appearance of ribosomal 28-S RNA into the cytoplasm is severely affected (85-90% inhibition), in much higher proportions than the formation of its immediate precursor, nuclear 28-S RNA, thus suggesting a particular sensitivity to a lack of methylation of the last stage of preribosome maturation (possibly the release to cytoplasm). Finally, if a normal level of methylation does not appear to be stringently required for the completion of ribosomal RNA maturation, our results indicate that the degree of RNA methylation can modulate the general efficiency of the maturation processess.

Amino Acids↗

PubMed1979

Beta-adrenergic potentiation of the increased in vitro accumulation of cycloleucine by rat thymocytes induced by triiodothyronine.

We have previously demonstrated that 3,5,3'-triiodothyronine (T(3)), whether administered in vivo or added to suspending media in vitro, promptly stimulates the in vitro accumulation of the nonmetabolized amino acids, alpha-aminoisobutyric acid, and cycloleucine (CLE) by thymocytes isolated from weanling rats. In these studies, we have examined the in vitro interaction between catecholamines and T(3) with respect to this effect. The previously reported enhancement of CLE accumulation in thymocytes by T(3) in vitro (1 muM) was confirmed. When added alone in concentrations ranging between 10 nM and 0.1 mM, the adrenergic agonists, epinephrine and norepinephrine, had no effect on CLE accumulation. At a concentration of 1 muM, isoproterenol, terbutaline, and phenylephrine were also without effect. However, the effect of T(3) was clearly potentiated by the concomitant addition of epinephrine, norepinephrine, and possibly isoproterenol, whereas terbutaline and phenylephrine were without effect. Neither basal nor T(3)-enhanced CLE accumulation was affected by the addition alone of the adrenergic blocking agents, propranolol (0.1 mM), phentolamine (10 muM), or practolol (0.1 mM). Nevertheless, the beta(1)- and beta(2)-antagonist, propranol, and the beta(1)-antagonist, practolol, blocked the increment in CLE accumulation produced by epinephrine; the alpha-antagonist, phentolamine, was without effect. The enhancement of CLE accumulation that occurred in the presence of T(3), with or without epinephrine, was seen to be a result of an inhibition of CLE efflux, because T(3) alone inhibited CLE efflux, and this effect was increased when epinephrine was also present. On the other hand, neither T(3) alone nor T(3) plus epinephrine appreciably altered the rate of inward transport of CLE. As judged from studies of the ability of thymocytes to exclude trypan blue, neither T(3) alone nor T(3) plus epinephrine either enhanced or impaired viability of cells during 3-h periods of incubation. Cell water content, measured with [(3)H]urea, was unaffected by T(3), either alone or in the presence of epinephrine. In confirmation of previous results, the stimulatory effect of T(3) on CLE accumulation was unaffected by concentrations of puromycin sufficient to inhibit protein synthesis by at least 95%, and the potentiating action of epinephrine on the response to T(3) was similarly unaffected. From these findings, it is concluded that the effect of T(3) to increase CLE accumulation by thymocytes in vitro, though itself independent of adrenergic mediation, is potentiated by beta(1)-adrenergic stimulation. This interaction appears distinctly different from other thyroid hormone-catecholamine interactions, in which thyroid hormones enhance physiological responses to catecholamines. Its mechanism remains unclear, but the properties of the T(3) effect, and possibly the interaction itself, suggest that T(3) enhances CLE accumulation by an action at the level of the cell membrane.

Adrenergic beta-Agonists↗

PubMed1978

[Intestinal transport of cycloleucine (author's transl)].

Cycloleucine transport across the epithelial cells of the turtle intestine has been studied. It was assumed that the epithelium constituted a single homogeneous compartment. The results obtained by tracer techniques show that the epithelium, in transport, comprises more than one compartment, a separate one, at least, for intestinal absorption and secretion processes. This heterogeneous character makes impossible to attribute a tissue concentration to the epithelium as a whole, so that the existence of an aminoacid accumulation cannot be affirmed.

Amino Acids↗