Indolic compounds affect tryptophan binding to rat hepatic nuclei.

This study evaluates the effects of indolic or indole-related compounds on binding of L-tryptophan (saturable, stereospecific and of high affinity) to rat hepatic nuclei or nuclear envelopes. Addition of any one of many indolic or indole-related compounds, and particularly of 3-methylindole, does not inhibit in vitro binding of [3H]tryptophan to hepatic nuclear envelopes. However, when 3-methylindole (10(-10) to 10(-4) mol/L) is added in combination with unlabeled L-tryptophan (10(-4) mol/L), it diminishes the inhibitory effect of unlabeled L-tryptophan alone. Scatchard analysis of the binding affinities of in vitro [3H]tryptophan binding to hepatic nuclear envelopes using L-tryptophan in the absence or presence of 3-methylindole reveals similar dissociation constants (KD) under the two conditions, but the binding concentrations (Bmax) were greater in the combined group compared with that in the L-tryptophan alone group. Addition of 3-methylindole to liver before homogenization decreases specific [3H]tryptophan binding to nuclei compared with controls (without addition). L-Tryptophan tube-fed to rats with or without 3-methylindole administration increases in vitro hepatic protein synthesis compared with that of saline tube-fed controls. 3-Methylindole itself does not affect protein synthesis. Our report describes the effects of 3-methylindole on specific tryptophan binding to hepatic nuclear envelope receptor and discusses the possible implications thereof.

Studies with 1,1'-ethylidenebis(tryptophan), a contaminant associated with L-tryptophan implicated in the eosinophilia-myalgia syndrome.

L-Tryptophan binds to a rat liver nuclear envelope protein, and this binding is saturable, stereospecific, and of high affinity. Utilizing an in vitro assay of [3H]tryptophan binding to rat hepatic nuclear envelopes, we have previously determined that the L-tryptophan obtained from Showa Denko and which was implicated in cases of the eosinophilia-myalgia syndrome (EMS) inhibited [3H]tryptophan binding differently than did control L-tryptophan (not implicated in EMS). Therefore, in this study we investigated whether the addition of 1,1'-ethylidenebis(tryptophan) (EBT), a contaminant or impurity in L-tryptophan implicated in EMS, would have an effect. Our results indicate that EBT alone has little inhibitory binding effect compared with that of control L-tryptophan and that when EBT was added to control L-tryptophan the inhibitory binding effort was similar to that of control L-tryptophan alone. On the other hand, in vitro addition of EBT plus L-tryptophan to nuclei of cultured murine macrophages (WLG5) results in less inhibition of [3H]-tryptophan binding than does addition of L-tryptophan alone. Similar in vitro additions to nuclei of rat brain reveal little effect on binding, as was also the case for hepatic nuclear envelopes. Adding EBT to an in vitro hepatic protein synthesis system and measuring [3H]tryptophan incorporation into acid-precipitable proteins reveal that it competes similarly to that found with equimolar concentrations of unlabeled L-tryptophan. It does not affect [14C]leucine incorporation into proteins. [14C]EBT becomes incorporated in vitro into proteins (acid-precipitable), and this incorporation is diminished in the presence of equimolar concentrations of unlabeled EBT or L-tryptophan. This suggests that EBT or possibly a breakdown product becomes incorporated into proteins. Speculation as to how EBT may affect tissues in experimental animals is presented.

Effect of 3-phenylamino-L-alanine on tryptophan binding to rat hepatic nuclear envelopes.

We have determined that the addition of 3-phenylamino-L-alanine (PAA), a recently reported contaminant in L-tryptophan implicated in the eosinophilia-myalgia syndrome, affects tryptophan binding by utilizing an in vitro measurement of 3H-tryptophan binding to hepatic nuclei or nuclear envelopes. PAA (10(-10) to 10(-4) M) diminishes the inhibitory effect of binding due to excess unlabeled L-tryptophan (10(-4) M). PAA alone has no inhibitory effect on binding. The effect of PAA on in vitro tryptophan binding is in contrast to that of another contaminant, 1,1'-ethylidenebis(tryptophan), which together with excess unlabeled L-tryptophan does not appreciably affect the binding. In vitro addition of PAA and L-tryptophan to nuclei of rat brain or of cultured murine macrophages does not affect [3H]tryptophan binding in comparison to L-tryptophan alone as is the case with hepatic nuclear envelopes. Adding PAA to an in vitro protein synthesis system and measuring [3H]tryptophan or [3H]alanine incorporation into acid-precipitable proteins reveals that it competes similarly, but somewhat less, than does equimolar concentrations of unlabeled L-tryptophan or L-alanine, respectively. This suggests that PAA or a breakdown compound becomes incorporated into proteins. Speculation as to how PAA may affect tissues in experimental animals is presented.

Tryptophan binding to nuclei of rat brain.

Nuclei purified from whole rat brain specifically bind [3H]tryptophan ([3H]Trp) under in vitro conditions. Excess unlabeled Trp (10(-4) M) is an effective inhibitor of in vitro [3H]Trp binding to brain nuclei. Rats tube-fed L-tryptophan (Trp) (30 mg/100 g body wt) 30 min to 4 hr before killing revealed decreased specific binding of [3H]Trp to purified brain nuclei in vitro. By Scatchard analysis, the nuclei from whole brain appear to contain one binding site for [3H]Trp, and the KD is 263 nM. A number of Trp-related compounds, Trp metabolites, or other amino acids and their analogues were observed to compete for in vitro [3H]Trp binding to brain nuclei. The ability of Trp analogues, metabolites, and other cognate compounds to inhibit in vitro [3H]Trp binding to brain nuclei was evaluated and utilized to map the active site of Trp binding.

Competitive studies relating to tryptophan binding to rat hepatic nuclear envelopes as a sensitive assay for unknown compounds.

Our laboratory has reported that L-tryptophan binds to a rat liver nuclear envelope protein and this binding is saturable, stereospecific and of high affinity. Utilizing an in vitro [3H]tryptophan binding assay to hepatic nuclear envelopes, we have determined the effects of using excess unlabeled L-tryptophan from a number of different suppliers. This study reports that, based on our in vitro binding assay, some significant differences were observed when implicated L-tryptophan in cases of the eosinophilia-myalgia syndrome obtained from a Japanese manufacturer, Showa Denko, was assayed, in contrast to non-implicated L-tryptophan from other suppliers. An isolated impurity of Showa Denko L-tryptophan, 1,1'-ethylidenebis(tryptophan) alone or together with non-implicated L-tryptophan or its breakdown product, 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid, did not appreciably affect the in vitro [3H]tryptophan binding to hepatic nuclear envelopes as did the Showa Denko L-tryptophan. Our data, derived with our in vitro binding assay system, suggests that implicated L-tryptophan from Showa Denko contains a compound/s (unknown at present) other than 1,1'-ethylidenebis(tryptophan), which alters in vitro [3H]tryptophan binding. The significance of the impurity/ies involved remains to be determined.

Inhibitory effect of demoxepam on tryptophan binding to rat hepatic nuclei.

Since some patients with eosinophilia-myalgia syndrome ingested tryptophan along with benzodiazepines, we investigated whether demoxepam, the N-desalkylated compound of chlordiazepoxide, would influence the binding of tryptophan to hepatic nuclei. L-Tryptophan has been shown to bind (saturable, stereospecific, and of high affinity) to rat hepatic nuclei and nuclear envelopes. We report that demoxepam has an inhibitory effect on in vitro [3H]tryptophan binding to rat hepatic nuclei and has an apparent KD approximately 22 microM.

Studies with compounds that compete with tryptophan binding to rat hepatic nuclei.

Tryptophan has been demonstrated to affect hepatic RNA and protein metabolism. Binding of tryptophan to nuclear envelope proteins has been demonstrated to be saturable, stereospecific, and of high affinity. The hepatic nuclear envelope tryptophan binding protein (glycoprotein) has been purified to apparent homogeneity using either concanavalin A-agarose or tryptophan-agarose. The receptor has an Mr of approximately 34,000, which is the same as that observed when [3H]tryptophan has been crosslinked to nuclear proteins. In this study, we investigated whether analogs, metabolites or related compounds of tryptophan as well as other amino acids may bind to rat hepatic nuclei using in vitro [3H]tryptophan binding assays. Our results indicate that compounds that compete with [3H]tryptophan binding to hepatic nuclei or nuclear envelopes contain the alpha-amino-propionic acid structure. Such compounds were 5-fluoro tryptophan, 7-aza tryptophan, 5-hydroxy tryptophan, alanine, phenylalanine, tyrosine, cysteine and cystine. It was of interest that, whereas tryptophan-methyl ester and tryptophan-ethyl ester competed, alpha-methyl tryptophan, N-formyl tryptophan, N-acetyl tryptophan, and N-methyl tryptophan did not compete with [3H]tryptophan binding to hepatic nuclei or nuclear envelopes. Nonetheless, only the in vivo administration of L-tryptophan was able to stimulate nucleocytoplasmic efflux of hepatic RNA and protein synthesis.

Effect of benzodiazepines on tryptophan binding to rat hepatic nuclei.

This study evaluates whether or not some of the benzodiazepines would influence the binding of L-tryptophan to rat hepatic nuclei or nuclear envelopes. Previous publications have indicated that binding of L-tryptophan to hepatic nuclear envelope proteins was saturable, stereospecific, and of high affinity. In this study, we investigated whether some of the benzodiazepines would influence L-tryptophan binding to rat hepatic nuclei or nuclear envelopes as assayed by in vitro L-(5-3H) tryptophan binding. Our results indicate that the addition of chlordiazepoxide, diazepam, prazepam, flurazepam, nordazepam, N-desalkylflurazepam, temazepam, oxazepam, lorazepam, or 4-chlorodiazepam has little influence on the L-(5-3H) tryptophan binding to hepatic nuclei in vitro. However, the addition of demoxepam, the N-desalkylated compound of chlordiazepoxide, caused marked competition with 3H-tryptophan binding to hepatic nuclei in vitro. When chlordiazepoxide (1 mg/100 g body weight) is administered intraperitoneally 20 min before killing, the isolated hepatic nuclei reveal decreased specific L-tryptophan binding compared to controls. Also, rats pretreated with chlordiazepoxide intraperitoneally before tube-feeding L-tryptophan revealed diminished tryptophan-induced hepatic nuclear RNA efflux and protein synthesis. Our results suggest that chlordiazepoxide, possibly by itself or through a metabolite, can act to affect hepatic nuclear binding of L-tryptophan and to inhibit the stimulatory effect of L-tryptophan on hepatic protein synthesis.

Selective elevation of c-myc transcript levels in the liver of the aging Fischer-344 rat.

The expression of several proto-oncogenes involved in normal cell growth was examined as a function of age in male Fischer-344 rats aged 3, 6-9, 12 and 21-23 months. Northern blot analysis using RNA isolated from pooled livers or brains showed that the transcript levels of c-myc, but not c-sis or c-src-related genes, were markedly elevated in the liver with age. In contrast, there was no substantial change in transcript levels of any of these genes in aging brain. When c-myc expression was analyzed in livers from individual rats ranging in age from 4 to 22 months, a significant (p less than 0.01) 5-fold increase in c-myc transcript levels in relation to age was detected. The results indicate that expression of c-myc in rat liver is modulated during aging and more generally, that aging in rats is associated with organ-specific changes in the transcript levels of particular genes.

Absence of age differences in protein synthesis by rat brain, measured with an initiating cell-free system.

A cell-free protein synthesis system was derived from brains of young (3 month) and old (greater than 23 month) male Fischer-344 rats in order to examine brain protein synthesis in relation to age. The system was shown to be capable of reinitiating protein synthesis in vitro, and of synthesizing protein from exogenously added mRNA. Optimal ionic conditions for amino acid incorporation were 200 mM potassium ion and 5 mM magnesium ion, and amino acid incorporation depended on addition of ATP, GTP, and an energy-generating system (creatine phosphate and creatine phosphokinase). Amino acid incorporation was sensitive to the initiation inhibitors aurintricarboxylic acid and sodium fluoride. Optimal conditions were independent of the age of the rat from which the brain was taken. There was no statistically significant relation (p greater than 0.05) between capacity of amino acid incorporation and age. The aggregation state of brain polyribosomes also did not differ between young (3 month) and old (30 month) rats. The results suggest that overall brain protein synthesis capacity is age-invariant in the rat.

Regional analysis of rat brain proteins during senescence.

Brains of male Fischer-344 rats aged 3-4 months and 28-30 months were dissected into 11 regions, and the patterns of brain proteins in these regions were analyzed using two dimensional gel electrophoresis in conjunction with a sensitive silver stain detection method. Several hundred abundant and moderately abundant brain proteins were detected in each region using this method. At 3-4 months, most proteins were present in approximately equal amounts in each of the 11 regions. On the whole, this distribution was maintained as a function of age. One protein of 21 kdal pI 5.1 was present in the cerebellum in greater amount than the other regions at both 3-4 months and 28-30 months. Two proteins, 44 kdal pI 5.4 and 47 kdal pI 5.2 were present at increased levels in the inferior colliculus of 28-30 month animals compared to 3-4 month animals. Of the abundant and moderately abundant brain proteins detected by this method, there were none which showed major decreases in levels as a function of age. These results provide support for the concept that the molecular mechanisms which result in differential gene expression in different regions of the young adult rat brain are operative and are maintained in the brains of senescent rats.

A soluble factor (less than 4000 Da) from chick spinal cord blocks slow hyperpolarizing afterpotentials in cultured rat muscle cells.

The slow hyperpolarizing afterpotential (slow HAP) following an evoked action potential in cultured rat muscle cells is inhibited by coculture with spinal cord neurons, spinal cord-conditioned medium (CM) and by a low-molecular-weight fraction (less than 4000 Da) prepared from this CM. The incidence, amplitude and duration of the slow HAPs decreased significantly after 24 h of incubation. In contrast, chick retinal neuron cocultures or CM did not alter the slow HAP.

Preparation of a cell-free extract from rat brain which can initiate protein synthesis in vitro.

A cell-free protein synthesis system, derived from brains of 3 mo-old male Fischer-344 rats, has been characterized. The optimum conditions for amino acid incorporation in the system were 5 mM magnesium ion and 200 mM potassium ion. Incorporation depended on the addition of ATP, GTP, and an energy-generating system, and was sensitive to addition of the drugs aurintricarboxylic acid and sodium fluoride, inhibitors of initiation of protein synthesis. Both 40S and 80S initiation complexes were labeled in vitro, using [35S]methionine. Such labeling was sensitive to the protein synthesis inhibitors, aurintricarboxylic acid and sodium fluoride. The system, which can initiate protein synthesis, should be of use for examining mechanisms which underlie alterations in rat brain protein synthesis induced by various treatments.

Effect of intravenous administration of D-lysergic acid diethylamide on initiation of protein synthesis in a cell-free system derived from brain.

An initiating cell-free protein synthesis system derived from brain was utilized to demonstrate that the intravenous injection of D-lysergic acid diethylamide (LSD) to rabbits resulted in a lesion at the initiation stage of brain protein synthesis. Three inhibitors of initiation, edeine, poly(I), and aurintricarboxylic acid were used to demonstrate a reduction in initiation-dependent amino acid incorporation in the brain cell-free system. One hour after LSD injection, there was also a measurable decrease in the formation of 40S and 80S initiation complexes in vitro, using either [35S]methionine or [35S]Met-tRNAf. Analysis of the methionine pool size after LSD administration indicated there was no change in methionine levels. Analysis of the formation of initiation complexes in the brain cell-free protein synthesis system prepared 6 h after LSD administration indicated that there was a return to control levels at this time. The effects of LSD on steps in the initiation process are thus reversible.

Synthesis of S100 protein on free and membrane-bound polysomes of the rabbit brain.

Free and membrane-bound polysomes were isolated from the cerebral hemispheres and cerebellum of the young adult rabbit. The two polysomal populations were translated in an mRNA-dependent cell-free system derived from rabbit reticulocytes. Analysis of the [35S]methionine-labeled translation products on two-dimensional polyacrylamide gels indicated an efficient separation of the two classes of brain polysomes. The relative synthesis of S100 protein by free and membrane-bound polysomes was determined by direct immuno-precipitation of the cell-free translation products in the presence of detergents to reduce nonspecific trapping. Synthesis of S100 protein was found to be twofold greater on membrane-bound polysomes compared with free polysomes isolated from either the cerebral hemispheres or the cerebellum. In addition, the proportion of poly-(A+)mRNA coding for S100 protein was also twofold greater in membrane-bound polysomes compared with free polysomes isolated from the cerebral hemispheres. These results indicate that the cytoplasmic S100 protein is synthesized predominantly on membrane-bound polysomes in the rabbit brain. We suggest that the nascent S100 polypeptide chain translation complex is attached to the rough endoplasmic reticulum by an ionic interaction involving a sequence of 13 basic amino acids in S100 protein.

Heat shock protein in mammalian brain and other organs after a physiologically relevant increase in body temperature induced by D-lysergic acid diethylamide.

A physiologically relevant increase in body temperature from 39.7 to 42.5 degrees C, which was generated after the intravenous injection of D-lysergic acid diethylamide (LSD), caused the induction of synthesis of a 74,000-dalton heat shock protein in the brain, heart, and kidney of the young adult rabbit. A marked increase in the relative labeling of a 74,000-dalton protein was noted after analysis of both in vivo labeled proteins and cell-free translation products of isolated polysomes. A temporal decrease in the synthesis of this protein was noted as LSD-induced hyperthermia subsided. The 74,000-dalton protein, which is induced in various organs of the intact animal at a body temperature similar to that attained during fever reactions, may play a role in homeostatic control mechanisms.

Translation of mRNA associated with monosomes and residual polysomes following disaggregation of brain polysomes by LSD and hyperthermia.

Intravenous administration of LSD to young adult rabbits induces a transient disaggregation of brain polysomes and a relocalization of mRNA from polysomes to monosomes. To analyze the spectrum of mRNA molecules which were associated with either the residual polysomes or the translationally inactive monosome complex, these two fractions were isolated on sucrose gradients and translated in a reticulocyte cell-free system. Analysis of [35S]methionine labeled translation products by one and two dimensional gel electrophoresis revealed that a full spectrum of mRNA molecules was relocalized from polysomes to monosomes following drug induced polysome disaggregation. The only exception was the mRNA coding for the LSD-induced 74K protein which was associated with the residual polysome fraction and not with the monosome complex. This brain protein is similar in molecular weight to one of the major 'heat shock' proteins which are induced in tissue culture cells following elevation of ambient temperature and disaggregation of existing polysomes. The mRNA coding for the 74K brain protein was not observed in polysomes isolated following blockage of LSD-induced hyperthermia but it was noted when hyperthermia was induced by elevation of ambient temperature. The mRNA species coding for the 74K protein was polyadenylated.

Cell-free translation of free and membrane-bound polysomes and polyadenylated mRNA from rabbit brain following administration of d-lysergic acid diethylamide in vivo.

Free and membrane-bound polysomes and polyadenylated mRNA isolated from rabbit brain were translated in an mRNA-dependent rabbit reticulocyte lysate system. Electrophoretic analysis of the cell-free translation products demonstrated that although most of the nascent proteins were common to both free and membrane-bound brain polysomes, qualitative and quantitative differences were observed. Compared with the results obtained with purified polyadenylated mRNA, the addition of intact polysomes to the cell-free translation assay was more efficient and produced higher molecular weight products. Analysis of the translation products of free and membrane-bound polysomes revealed the appearance of 74K protein following neither LSD administration or hyperthermia induced by elevated temperature treatment. The presence of this 74K protein was verified by analysis of the translation products by two-dimensional gel electrophoresis.

Characterization of an initiating cell-free protein synthesis system derived from rabbit brain.

Protein synthesis in the brain is known to be affected by a wide range of treatments. The detailed analysis of the mechanisms that are involved would be facilitated by the development of cell-free translation systems derived from brain tissue. To date, brain cell-free systems have not been fully characterized to demonstrate a capacity for initiation of translation. The following criteria were utilized to demonstrate that a cell-free protein synthesis system derived from rabbit brain was capable of initiation in vitro: (a) sensitivity of cell-free translation to the initiation inhibitor aurintricarboxylic acid (ATA); (b) binding of [35S]Met-tRNAf to 40S and 80S initiation complexes; (c) incorporation of labeled initiation methionine into high-molecular-weight proteins; and (d) the association of labeled exogenous mRNA with polysomes. The optimum conditions for amino acid incorporation in this system were 4 mM-Mg2+, 140 mM-K+, and pH 7.55. Incorporation was dependent on the addition of ATP, GTP, and an energy-generating system. Cell-free protein synthesis reflected the normal process, since a similar spectrum of proteins was synthesized in vitro and in vivo. This initiating cell-free translation system should have wide application in the analysis of the mechanisms whereby various treatments affect protein synthesis in the brain.

Effect of intravenous administration of d-lysergic acid diethylamide on subsequent protein synthesis in a cell-free system derived from brain.

An initiating cell-free protein synthesis system derived from brain was utilized to demonstrate that the intravenous injection of d-lysergic acid diethylamide (LSD) to rabbits induced a transient inhibition of translation following a brief stimulatory period. Subfractionation of the brain cell-free system into postribosomal supernatant (PRS) and microsome fractions demonstrated that LSD in vivo induced alterations in both of these fractions. In addition to the overall inhibition of translation in the cell-free system, differential effects were noted, i.e., greater than average relative decreases in in vitro labeling of certain brain proteins and relative increases in others. The brain proteins of molecular weights 75K and 95K, which were increased in relative labeling under conditions of LSD-induced hyperthermia, are similar in molecular weight to two of the major "heat shock" proteins reported in tissue culture systems. Injection of LSD to rabbits at 4 degrees C prevented LSD-induced hyperthermia but behavioral effects of the drug were still apparent. The overall decrease in cell-free translation was still observed but the differential labeling effects were not. LSD appeared to influence cell-free translation in the brain at two dissociable levels: (a) an overall decrease in translation that was observed even in the absence of LSD-induced hyperthermia and (b) differential labeling effects on particular proteins that were dependent on LSD-induced hyperthermia.