NUTRITION REVIEWS VOL. 21 JUNE 1963

SOME THOUGHTS ABOUT THE y-AMINOBUTYRIC ACID SYSTEM IN NERVOUS TISSUE

The subject to be surveyed briefly here is of interest to workers in several disci- plines because i t has become one of the biological areas in which biochemistry, pharmacology, electron microscopy, and electrophysiology have begun to meet a t the level of the nerve cell in an attempt to explain some aspects of its unique function. Work in our laboratory and in many others is concerned with studies of the metabolic and physiological relationship of y-amino- butyric acid (yABA) , an easily extractable, simple substance with a unique occurrence in the central nervous system (CNS) of vertebrate organisms, in which it is found in extracts of brain, spinal cord, and retina [see pertinent reviews by many impmtant contributors to this work in Inhibition in the Nervous System and 7-Aminobutyric Acid, E. Roberts e t al., Eds., Pergamon Press, Oxford, 1960; E. Roberts and E. Eidelberg, International Review of Neuro- biology 2, ,279 (1960) ; K. A. C. Elliott and H. H. Jasper, Physiol. Rev. 39, 383 (1959)l.

In general, gray matter contains much more yABA than white matter. Of the vari- ous areas of brain studied quantitatively the levels of YABA, but not of the other free amino acids, are outstandingly high in the hypothalamus, an area of brain known ta contain remarkably large amounts of several neuro-active substances, Until re- cently yABA has beeh found in relatively high levels in the CNS of all invertebrates studied, including insects, and in both the peripheral nervous system and the CNS in crabs and lobsters. However, we now have observed that yABA and the L-glutamic acid decarboxylase (GAD), the enzyme which catalyzes the formation of yABA

No. 6

from L-glutamic acid, are present in only barely detectable levels in the octopus brain. It is possible that a comparison of the types of cells in the octopus nervous system with those in species which contain high levels of the yABA system might give valuable suggestions with regard to the morphological elements with which this system is associated.

yABA is formed in the CNS to a large extent, if not entirely, from L-glutamic acid. The reaction is catalyzed by GAD, an enzyme found in mammalian organisms only in the CNS, largely in the gray mat- ter. The reversible transamination of YABA with a-ketoglutarate is catalyzed by a transaminase (yABA-T) , which in the CNS is found chiefly in the gray matter, but also is found in other tissues. Both GAD and yABA-T are B6 enzymes. The products of the transaminase reaction are succinic semi- aldehyde and glutamio acid. If a ready metabolic source of s:, ccinic semialdehyde were available, yABA could be formed by the reversal of the rlaction. However, to date no convincing eTiidence has been ad- duced for the formation of significant amounts of yABA by reactions other than the decarboxylation of L-glutamic acid.

Brain also contains a dehydrogenase which catalyzes the oxidation of succinic semialdehyde to succinic acid, which in turn can be oxidized via the reactions of the tri- carboxylic acid cycle. Consistent with these relationships, glutamic acid, yABA, and succinic semialdehyde can be oxidized by various brain preparations and can support oxidative phosphorylation. Therefore, in the CNS, but not in other tissues, there can exist a metabolic shunt around the a-keto- glutarate oxidase system of the tricar-

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162 NUTRITION REVIEWS [Vol. 21, No. 6

boxylic acid cycle, the operation of which depends on the unique occurrence of GAD and yABA in the CNS, a formulation con- sistent with both in vitro and in vivo stud- ies performed to date.

Determinations of yABA levels in vari- ous cerebellar and cerebral cortical layers have shown the amounts of yABA to corre- late well with the distribution of enzymes involved in general energy metabolism and an estimate of approximately 40 per cent has been made for the contribution of the “yABA shunt” to oxidative metabolism of brain slices. Histological correlations with measurements of enzyme activity have sug- gested that both GAD and yABA-T may be associated with neuronal elements in the CNS of mammals, but there is still no con- clusive proof that the enzyme activities are not in the glial elements which are inti- mately associated with the neurons in the areas studied. Unfortunately, reliable histo- chemical localization of yABA and GAD or yABA-T activities has not yet proved feasible. The enzymes involved in the “yABA shunt” even may not be present in the same location in the CNS. Recent ex- periments have shown that upon adminis- tration to animals of hydroxylamine or aminooxyacetic acid, substances which are potent in vitro inhibitors of both the GAD and the yABA-T in brain, only the trans- aminase was inhibited and there were marked elevations of yABA content in the brains of the treated animals. One of the simplest possibilities is that the two en- zymes are present in different cell types or in different intracellular sites in the same cells in the CNS and that the above car- bony1 reagents penetrate to the regions con- taining the transaminase, but not to those wherein the decarboxylase is located. The possibility of the presence of the enzymes in different types of cells also is suggested by the failure to find a correlation between GAD and yABA-T levels in various cere- bral areas. Progressive increases in yABA

content with age during development and of the activities of GAD and YABA-T have been found in all species studied to date.

There is a remarkable constancy in the concentrations of easily extractable ninhy- drin-reactive substances found in the tis- sues of mature animals under a variety of physiological conditions, the characteristic distributions of most of these constituents being largely maintained even when major disturbances are produced in the homeo- static mechanisms of the animal as a whole by a variety of procedures which include alterations in endocrine balances, dietary deficiencies of various types, starvation, de- hydration, tumor growth, and the injection of effective doses of a large variety of drugs and antimetabolites (E. Roberts and D. G. Simonsen in Amino’ Acids, Proteins, and Cancer Biochemistry, J . T . Edsall, Editor, p . 121. Academic Press, N e w York, 1960). This suggests that the concentrations of most of these substances are regulated largely by metabolic servomechanisms which not only coordinate the rates of mul- tiple biosynthetic and degradative utilizing pathways, but also which adjust continu- ally the rates of entry into cells and exit therefrom.

It is feasible to study some of the fact,ors regulating levels of yABA because there ap- pears to be only one major mode of forma- tion in brain and one chief pathway of utilization. yABA is not an inhibitor of GAD and, therefore, does not exert a feed- back inhibition of its formation from glu- tamic acid. Problems related to movement between cells and extracellular fluid also are minimal since parenterally administered yABA does not pass the blood-brain barrier readily in normal mature animals, and the present evidence favors the view that yABA, once formed in brain or injected into it, cannot leave the brain readily and enter the blood.

From the above it would appear that the yABA levels in the various parts of the

June 19631 NUTRITION REVIEWS 163

CNS must be largely a function of the rela- tive rates of formation and utilization by the GAD and YABA-T pathways, respec- tively, and of the retention of yABA by the tissue in which i t is formed. Although the activities of the above two enzymes may be dependent on many factors, the property of these enzymes which has proven most useful in studying the regulation of yABA levels in vivo is their requirement for pyridoxal phosphate as a coenzyme. It has been possible to devise methods of decreas- ing or increasing levels of yABA by differ- ential inhibition of these enzymes in vivo. Some of the agents that have been found to inhibit the enzymes act by decreasing the amount of pyridoxal phosphate available for association with the apoenzymes, others inhibit enzymatic activity by reacting with the enzyme-bound coenzyme, and still others can do both. It is not surprising that the quantity of glutamic acid, the precursor of yABA in brain, remains essentially un- changed whether the amount of yABA is increased by blocking its utilization with hydroxylamine or aminooxyacetic acid or decreased by inhibiting its formation with thiosemicarbazide or 4-methoxymethylpyri- doxine.

In contrast to yABA, there are several modes of glutamate metabolism in brain. A decreased rate of utilization by one could be compensated for by increased rates of utilization along alternate pathways or by a decreased rate of formation. The servo- mechanisms for the control of glutamate levels, and the amounts of most other easily extractable ninhydrin-reactive constituents found in brain, appear to be more versatile than those for yABA.

Although physiological findings with var- ious test systems, ranging from crayfish stretch receptor to the monkey cortex, have suggested strongly that yABA has inhibi- tory properties and that endogenous levels of yABA may be involved in some aspects of the control of neuronal excitability in

the CNS, recent observations have empha- sized the degree of caution which must be exerted in interpreting correlations ob- tained between measurements of yABA levels and such phenomena as gross con- vulsive seizures in animals or electrical re- cordings obtained from various brain or spinal cord structures. No simple and direct relationship has been found by us between electrical excitability and the level of yABA normally found in particular areas of cat brain cortex. It is obvious that the degree of activity in any part of the CNS must be a result of the multiple interactions be- tween inhibitory and excitatory influences. At present all of these influences are not known and i t is not possible to evaluate all of the known factors even under particular, highly controlled experimental circum- stances.

yABA is capable of existing in nervous tissue in free and bound forms, but ade- quate techniques have not yet been devised for the accurate quantitative assessment of the amount of free and bound yABA in a particular area of brain. It is not known which form is more significant from the physiological point of view. Data obtained to date in our laboratory on the binding of yABA are similar to some of the findings made previously on a number of substances active in the CNS, such as acetylcholine, the catecholamines, 5-hydroxytryptamine, and histamine.

Thus, i t appears that a t least some of the endogenous yABA may be associated with sedimentable particulate subcellular fractions which are found by electron mi- croscopy to be rich in structures of a vesic- ular and membranous nature. Exogenously added labeled yABA can be bound in a nonenzymatic fashion by material found in the same gross centrifugal fractions by a structure which is osmotically sensitive and which has properties expected of a macro- molecular complex containing lipid, pro- tein, and possibly polysaccharide. The lat-

164 NUTBITION REVIEWS [VOZ. 21, NO. e

ter type of binding of yABA is not observed in any tissue other than that of the CNS. A high degree of specificity appears to exist a t the yABA binding sites, since compounds closely related in structure to yABA are only relatively poor inhibitors of the bind- ing of yABA. A remarkable feature of the yABA binding system is a specific require- ment for Na+ ions.

Although convulsive seizures have been produced in animals by treatment with var- ious vitamin Be antimetabolites or with some carbonyl-trapping agents which also reduce yABA levels in brain, these sub- stances may have many additional physio- olgical effects resulting in alterations in cerebral excitability, changes in the perme- ability of the blood-brain barrier, and di- rect or indirect inhibitions of many en- zymes which do not require pyridoxal phosphate as a coenzyme. This was illus- trated forcefully in experiments in which i t was shown that thiosemicarbazide which, when given by itself inhibits GAD activity and lowers yABA levels while producing seizures, when injected simultaneously with hydroxylamine invariably causes seizures in rats a t a time when the quantities of yABA are elevated or remain normal in all areas of brain studied.

Observations in many laboratories have established a relationship of convulsive seizures to dietary pyridoxine deficiency in experimental animals and in human beings. In pyridoxine deficient rats there was found to be a decrease of approximately 50 per cent in the degree of the saturation of the apoenzyme of GAD with pyridoxal phos- phate, the content of apoenzyme remaining normal. However, definitive studies have not been performed on the effects of pro- gressive, simple dietary deficiency of the vitamin on yABA levels and on the func- tion of the related enzymes in vivo as well as on other chemical systems dependent on pyridoxal-phosphate requiring enzymes which are known to have important func- tions in neuronal tissues such as those in-

volving the catecholamines and serotonin. Perhaps such studies will offer the best op portunities to identify the system which be- comes rate-limiting when neuronal control mechanisms break down in vitamin Be de- ficiency.

Nearly all of the physiological observa- tions have shown yABA to be inhibitory, a greater amount of a given type of stimula- tion being required to elicit the same re- sponse from a particular neuronal element after application of yABA than before. Re- sults from both vertebrate and invertebrate test systems suggest that one of the prob- able common modes of action of neural in- hibition (postsynaptic), in general, and of exogenously applied yABA is to cause an increase in neuronal membrane conductance which is attributable to an increased flow of K+ and/or C1- ions. yABA mimics the effects of the naturally-released inhibitory transmitter substance when applied both to peripheral and central synapses in crus- tacea. However, i t seems generally agreed that the physiological observations on the action of yABA, together with the finding of higher concentrations and lower rates of turnover of yABA in the nervous system than for substances like acetylcholine sand the catecholamines, make it seem unlikely that yABA, itself, is the classically defined inhibitory transmitter in the vertebrate nervous system. However, the possibiliQ that yABA may have effects on the release of inhibitory and excitatory transmitters from presynaptic endings and thus modify neuronal excitability is an intriguing one.

There is great intrinsic interest in under- standing the role of yABA, an inhibitory substance which seems to be formed and located entirely in the CNS, largely in the gray matter, in the various vertebrate spe- cies studied. The situation is a favorable one today because we are dealing with rela- tively large quantities of a chemically stable substance for the determination of which there are elegant micro-analytical methods. Subtle electrophysiological tech-

June 19631 NUTRITION REVIEWS 165

niques can monitor the effect6 of yABA on individual cells and powerful electron mi- croscopic methods can help with the locali- eation of subcellular structures in which Chairman portions of the yABA system may be lo- cated. There is reason to hope that within the next few years it will be possible to

define the role of the yABA system at a molecular level in specific cells in the CNS.

EUGENE ROBERTS, PH.D.

Department o j Biochemistry City of Hope Medical Center Duarte, California

FLUORIDE, BONE CRYSTAL STRUCTURE, AND CALCIUM BALANCE

With increasing fluoride concentrations in human bone, increased “crystallinity” as evidenced by larger crystal size and more nearly perject crystals has been observed. Early studies indicate an indirect relationship between fluoride and carbonate con- centrations in the enamel and a direct relationship between fluoride ingestion and calcium retention.

The linear relationship between the level of fluoride in the drinking water and the concentration of fluoride in human iliac crest, rib, and vertebra has been reviewed (Nutrition Reviews 19,124 (1961)). In the same investigation, the authors observed that, as bone fluoride increased, a substan- tial decrease in carbonate, a striking de- crease in citrate, a slight but consistent de- crease in sodium, and a minor generalized decrease in potassium concentration oc- curred. The authors postulated that these relationships might reside in the possible surface orientation of carbon dioxide, cit- rate, sodium, and potassium on the apatite bone crystals.

In a recent investigation the same bone samples have been subjected to x-ray dif- fraction t o determine if the size and degree of perfection of the apatite crystals varied with the concentration of fluoride (I. Zip- kin, A. s. Posner, and E. D. Eanes, Bio- chim. Biophys. Acta 59,965 (1968) ) . X-ray diffraction patterns were taken of finely powdered dry fat-free bone samples which contained up to 1.0 per cent fluoride on an ash basis. The degree of resolution of the four principal x-ray reflections of the bone apatite pattern was used as a measure of the “crystallinity” of each sample.

The term “crystallinity,” as used, in-

cluded the effect of the size of the apatite crystals as well as the relative imperfection of the crystals since both parameters con- tribute to the broadening of x-ray diffrac- tion maxima. As crystal size and/or perfec- tion increased, “crystallinity” increased. In general the “crystallinity” of the bone apa- tite increased as the fluoride content of the iliac crest, rib, and vertebra increased. Analysis of covariance showed that this re- lationship was highly significant for iliac crest, rib, and vertebra (P < 0.001).

The authors noted that there were major variations in the “crystallinity’7 index a t each of the various fluoride levels and es- pecially a t the lower fluoride concentra- tions. These variations suggested that other variables than fluoride might be involved. Two graphic figures were presented of x- ray diffraction patterns for samples of iliac crest with fluoride levels of 0.224 and 0.873 per cent on an ash basis. The differences between these figures provided impressive visual evidence of the influence of fluoride concentration upon bone crystal structure.

As the concentration of fluoride acquired during bone growth and remodeling in- creased, the changes in crystal size and crystal imperfection would reduce the ef- fective surface area of the crystals in a given weight of bone, reduce the reactivity