Stanley Samuels

Transport of the large-neutral amino acids by the γ-glutamyl cycle: A proposal

Abstract The γ-glutamyl cycle has been proposed by Meister (1973) as one possible mechanism for the mediation of amino acid transport. The high energy requirement of the pathway, the very low specificity of γ-glutamyl transpeptidase and the inability to account for trans membrane stimulation of amino acid entry are but three criticisms of this hypothesis. It is proposed that the various objections can be overcome by postulating that the soluble form of γ-glutamyl transpeptidase transfers the γ-glutamyl moiety from gluthathione to glutamine (in the case of brain) and that the membrane sequestered form of this enzyme catalyzes the exchange of the γ-glutamyl group between γ-glutamyl glutamine and an entering neutral amino acid. The released glutamine leaves the cell. The γ-glutamyl amino acid then passes into the cytoplasm where it is acted upon by either γ-glutamyl cyclotransferase or the soluble γ-glutamyl transpeptidase which transfers the γ-glutamyl group to another molecule of glutamine. It is postulated that access to the membrane-bound enzyme is dependent on the relative lipophilia of the entering large-neutral amino acids. The available data support this mechanism. By regeneration of γ-glutamyl glutamine, a low expenditure of energy is required for the transport process. Specificity of transpeptidation is attained by the constraints of access to the membrane bound enzyme site.

Effect of γ-glutamyl cycle inhibitors on brain amino acid transport and utilization

Two inhibitors of the γ-glutamyl cycle, methionine sulfoximine (MSO) and 2-imidazolidone-4-carboxylic acid (ICA) were administered to C57BL/6J mice. Both agents resulted in a reduced rate of transport of tyrosine from blood to brain and a decreased rate of incorporation of tyrosine from plasma into brain protein. MSO administration also diminished the concentrations of brain tyrosine, dopamine, and norepinephrine. MSO decreased the transport rate of valine by brain as well as the rate of its incorporation into protein when expressed in relation to the plasma specific activity. The results demonstrate a significant role for the γ-glutamyl cycle in the transport of large neutral amino acids from blood to brain.

Age related changes in blood-to-brain amino acid transport and incorporation into brain protein

Blood-to-brain amino acid transport consists of at least two components: 1. a fast rate or early process, commonly measured by the intra-carotid bolus injection method and attributed to transport across the capillary endothelium and entry into the astrocytes, and, 2. a slow rate or later component measured over 2 to 15 minutes probably associated with exit from the astrocytes and entry into the neurons. Incorporation into brain protein is temporally related to the second process. In the present study the slow and fast rate transport components and the incorporation into brain protein of tyrosine (Tyr) and Valine (Val) was measured in young adult and aged male C57BL/6 mice. The results indicate that the fast rate transport component is unaffected by age while the rates of the slow process and protein turnover show an exponential decline most marked between 3 and 8 months of age. Changes in the relative incorporation of Tyr and Val suggest that brain protein metabolism is altered qualitatively as well as quantitatively in aging, in these animals.

CORRELATION OF GLIOMA CELL REGRESSION WITH INHIBITION OF INSULIN-LIKE GROWTH FACTOR 1 AND INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-2 EXPRESSION

To explore the antitumor effect of insulin-like growth factor 1 (IGF-I) antisense RNA and the interaction of IGF-I with insulin-like growth factor-binding proteins (IGFBPs) in glioma cells, a recombinant retrovirus expressing IGF-I antisense RNA was constructed and introduced into C6 glioma cells. IGF-I antisense RNA reverses the transformed phenotype in glioma cells and inhibits glioma cell growth by blocking overexpression of endogenous IGF-I. Expression of IGFBP-2 is increased in glioma cells as compared with normal adult glial cells. IGF-I antisense RNA also inhibits expression of IGFBP-2 in glioma cells, but does not influence expression of the other IGFBPs. Although IGFBP-2 in conditioned medium from wild-type C6 cell cultures itself does not directly influence glioma cell growth, it synergistically enhances exogenous IGF-I-mediated DNA synthesis in IGF-I-negative C6 cells. These findings indicate the inhibitory effect of IGF-I antisense RNA on growth and development of glioma cells. IGF-I-dependent glioma cell growth may, in some circumstances, require IGFBP-2 as a cofactor. The antitumor effect of IGF-I antisense RNA is also associated with inhibition of IGFBP-2 expression.

5-Fluorocytosine-mediated apoptosis and DNA damage in glioma cells engineered to express cytosine deaminase and their enhancement with interferon

To explore the antitumor mechanism of bacterial cytosine deaminase plus 5-fluorocytosine (CD/5-FCyt) in combination with interferons (IFNs), glioma cells were transduced with recombinant retroviruses expressing CD. The transduced glioma cells become sensitive to the nontoxic prodrug 5-FCyt. Apoptosis, DNA damage, bystander effect, and inhibition of thymidylate synthase (TS) and DNA synthesis are associated with CD/5-FCyt-mediated glioma cell killing. Furthermore, IFNs enhance this effect by increasing DNA damage and further inhibiting TS activity. The bystander effect is mediated by the release of cytotoxic metabolites of 5-FCyt into the extracellular milieu triggering apoptosis and DNA damage. Our data indicate that the use of CD/5-FCyt in combination with IFNs may provide a more effective approach for the treatment of brain tumors.

Anticonvulsant Activity of Glycylglycine and δ-Aminovaleric Acid: Evidence for Glutamine Exchange in Amino Acid Transport

We have proposed that glutamine serves in a facilitated diffusion process, mediated by the enzyme γ‐glutamyl transferase (γ‐glutamyl transpeptidase; γGT) and that it leaves the brain in exchange for entering amino acids. Glutamine is also a precursor of γ‐aminobutyric acid (GABA). Thus, providing an alternate substrate for γGT should spare brain glutamine, raise GABA, and cause an anticonvulsant effect. We have found that glycylglycine, the best‐known substrate for γGT, and δ‐aminovaleric acid (DAVA), a structural analog, have anticonvulsant activity in DBA/2J mice. Both compounds can decrease the incidence and severity of seizures induced by l‐methionine‐rs‐sulfoximine or electroconvul‐sive shock. DAVA was also tested and found to be active against seizures caused by pentylenetetrazol or picrotoxin. [14C]DAVA entered the brain at the rate of 18.7 nmol/g/min. The activity of DAVA as a substrate of γGT was intermediate to that of glycylglycine and glutamine. Preliminary studies have shown that brain glutamine and perhaps GABA are elevated 3 h after administration of DAVA (7.5 mmol/kg). These findings support the theory that glutamine exchange plays a role in amino acid transport across the blood‐brain barrier and suggests a new concept in anticonvulsant therapy. Key Words: Amino acids, transport—γ‐Aminobutyric acid—δ‐Amino valeric acid—Anticonvulsant—Glutamine, exchange—Seizures.

Procedure for measurement of amino acid transport from blood to brain in small animals

Abstract The exponential plasma specific activity curve 2.5 to 12.5 min after injection (sc) of [14C]tyrosine was integrated and divided by time to obtain the mathematical relationship between the average equivalent specific activity S and the measured specific activity S in any individual animal. S is the constant, average value of S that is equivalent to the curvllinearly varying quantity that the body tissues are actually exposed to. Dividing the total brain radioactivity by S gave the tissue Tyr uptake U. The function dU dt is linear from 2.5 to 12.5 min and represents the rate of uptake of the amino acid. Incorporation into protein was similarly measured. Brain uptake of Tyr averaged 7.06, and the apparent protein incorporation was 1.99 nmol/g of brain per min. The γ-glutamyl cycle inhibitor l -methionine-RS-sulfoximine reduced total brain uptake of tyrosine by 42.8% and the apparent rate of protein incorporation by 39.0%.

Direct quantification of micro-thin-layer chromatograms

Abstract A variety of compounds have been quantified after thin-layer electrophoresis and/or thin-layer chromatography on microplates by means of the triple product of the largest and smallest diameters and the maximum absorbance of each spot. The diameters were measured with a graduated magnifier and spot absorbance was determined by means of a fiber optic light pipe fitted to a standard spectrophotometer. Precision and accuracy of ± 5% or less were obtained using amino acids, glucose, and cholesterol in the range of 10 −11 −10 −9 moles. A total of six different reagents were used on silica gel and cellulose, with glass plates and Mylar film supports after both mono-dimensional and two-dimensional separations. Except for the lipids which were applied with a modified syringe, all solutions were spotted by means of calibrated Nichrome wire loops delivering 36–126 nl. The procedure was found to be simple and economical and reliable in routine use.

Amino acid transport inhibition: Brain and behavioral correlates

In vivo inhibition of uptake 14C-L-valine by brain following subcutaneous administration of either of two gamma-glutamyl cycle enzyme inhibitors, 2-imidazolidone-4-carboxylic acid (ICA), or, L-methionine-S-sulfoximine (MSO) is documented in C57BL/6J mice. Dose related decrease in exploratory activity, impairment of memory for foot shock, and reduced operant responding for food reinforcement parallels the time course for interference with uptake of a large neutral amino acid by these two compounds previously shown to inhibit different enzymes in the gamma-glutamyl cycle subserving active amino acid transport.

Rapid quantitative spotting of submicroliter volumes for thin-layer chromatography

Simple, inexpensive applicators for thin-layer chromatography can be easily constructed to reproducibly deliver 40 nl or less. They are appropriate for quantitative work in the 10−10 molar range with a coefficient of variation of less than 5%. Although on calibration the variability appears to be much greater, in incremental spotting the deviations from the mean are additive and the pluses and minuses cancel out. Two spotter designs are described one of which is made from Nichrome wire and the other from ordinary sewing needles.