Leon T. Kremzner

POLYAMINE METABOLISM IN THE CENTRAL AND PERIPHERAL NERVOUS SYSTEM

The complexity of the nervous system tissue relative to other tissues of the organism is such that it is in some respects less suitable for biochemical study; however, because of its unique character, the study of the nervous system may provide some special insight into both the metabolism and function of the polyamines. The neuron is not only one of the most actively biosynthesizing cells in the animal body, but it also, along with its supporting cells, provides the means for communication, learning and memory. In 1924, Dudley and coworkers isolated spermine from bovine brain; this was the first indication that the polyamines were present in the nervous system.l Subsequently, Hamalainen determined the spermine content of some discrete areas of the humain brain.? With the development of improved analytical procedures, employing ion exchange chromatography, Rosenthal and Tabor established the presence of both spermidine and spermine in the rat brain.3 This was followed by a report by Kewitz, in 1959, of the occurrence of putrescine and spermidine in pig brain.4 Subsequently, it was reported that S-adenosylmethionine, a precursor of both spermidine and spermine was present in, and synthesized by, the rat brain.5 The first information on the distribution of the polyamines in discrete regions of the nervous system was the result of a study of the rabbit brain by Shimizu and associates in 1964.G These investigators also noted marked differences in the polyamine concentration in gray and white matter of human brain cortex. More recently, putrescine has also been isolated from the human brain.7 Although the polyamines and their metabolic precursors are present in relatively large amounts in the central nervous system (CNS), quantitatively the polyamines are the major amine constituents, there is no definite knowledge of their biological function. Our studies are directed toward clarification of polyamine metabolism and function in the nervous system.

Metabolism of polyamines in mouse neuroblastoma cells in culture. Formation of gaba and putreanine.

—Polyamine metabolism of mouse neuroblastoma cells grown in culture was studied with special reference to the synthesis of GABA from putrescine and putreanine from spermidine. This study shows that neuroblastoma cells in the presence of a complete culture medium containing calf serum readily metabolized [14C]putrescine to GABA; the rate of synthesis is similar to the rate of synthesis of spermidine from putrescine. In the absence of serum the conversion of putrescine to GABA is minimal. In the presence of serum GABA formation is completely inhibited by the diamine oxidase inhibitor aminoguanidine. GABA synthesis does not occur in the absence of cells. The GABA synthesized is not readily metabolized to succinate or homocarnosine. Mouse neuroblastoma cells metabolized [14C]ornithine to putrescine, GABA, and spermidine. Spermidine was metabolized to putrescine, putreanine and spermine.

Electron Microscopic-Cytochemical and Biochemical Studies of Acetylcholinesterase Activity in Denervated Muscle of Rabbits

Acetylcholinesterase (AChE) activity has been studied in normal, control and denervated muscle of rabbits by electron microscopic-cytochemistry and radiometric assay. A small amount of butyrylcholinesterase (BuChE) activity is also found in biochemical assay of unfixed muscle, but it is not demonstrable cytochemically in fixed specimens by the method used in this study. Both a soluble and particulate AChE activity are present in all specimens examined. The particulate activity is probably due to enzyme localized in the sarcotubular system and at the motor end-plate. Soluble AChE activity may represent those sites exhibiting random cytochemical end product, such as some areas of normal and denervated muscle and muscle nuclei, Schwann cells, and AChE-containing mononuclear cells in the connective tissue. There is a greater proportion of particulate than soluble AChE activity in normal and control muscle, a finding which is compatible with the well localized cytochemical sites. Four to six weeks post-denervation, there is a marked increase in extrajunctional AChE activity to peak values 15 to 30 fold above control values. The increase is accompanied by a reversal in the proportion of particulate to soluble enzyme, so that there is almost twice as much soluble as particulate AChE. There are also numerous “spots” of random cytochemical end product throughout extrajunctional muscle. The increase in levels of AChE activity, the change to predominantly soluble form, and the large numbers of new cytochemically active sites indicate that synthesis of new enzyme has taken place. Changes in AChE activity in denervated rabbit have been compared to those occurring in dystrophic mouse muscle. It has been suggested that there might be a relationship between the formation of new extrajunctional sarcoplasmic sites of AChE activity and the spread of α-bungarotoxin binding sites and chemosensitivity in developing and denervated muscle.

Effects of some cholinesterase inhibitors on the squid giant axon. Their permeability, detoxication and effects on conduction and acetylcholinesterase activity.

Abstract The irreversible cholinesterase inhibitors, O , O -diisopropyl S-(2-diisopropyl-aminoethyl) phosphorothioate (Tetriso), O -ethyl Se-(2-diethyiaminoethyl) ethylphosphonoselenoate (Selenophos) and O , O -diethyl S-(2-ethylthioethyl) phosphorothioate (Isosystox), penetrate in their inhibitory forms into the squid giant axon. Tetriso and Selenophos, when applied for 30 min in concentrations as high as 5 × 10 −3 M, did not block axonal conduction. At that concentration, Isosystox caused either a reversible or an irreversible block of conduction depending on the length of time the nerve was exposed to the inhibitor. After pretreatment of the squid axon with cottonmouth moccasin venom, Selenophos (5 × 10 −3 M) caused in 25 min a marked and irreversible reduction in the action potential, while Tetriso (5 × 10 −3 m) caused a rapid and reversible decrease in the action potential. An extremely sensitive, simple radiometric technique for the assay of cholinesterase activity has been developed and is described in detail. Nevertheless, attempts to measure enzyme activity in squid axons exposed to Selenophos or Tetriso were unsuccessful because small amounts of entrapped inhibitor in the tissue were released upon homogenization. Possible explanations for the high concentrations of inhibitors required to affect conduction are discussed.

Chronic Cerebellar Stimulation in the Monkey: Preliminary Observations

In a single monkey, the surface of the cerebellum was stimulated electrically for 205 hours with electrodes and parameter values similar to those currently used in humans for treatment of epilepsy. Im pedance of stimulating and nonstimulating control electrodes remained unchanged throughout an observation period of six months. Potentials evoked by cerebellar stimulation could be recorded from the cranium, providing a noninvasive technique of determining the level of current delivered to cerebellum. Examination of the implantation site showed marked meningeal thickening surrounding the stimulating electrodes. Such thickening was not observed surrounding a control set. Light and electron microscopical examination revealed severe loss of Purkinje cells in tissue near the stimulating electrodes. There was also a moderate loss in other parts of cerebellar cortex down to a depth of about 1 mm from the exposed surface. Biochemical analysis revealed metabolic abnormalities consistent with the morphologic evidence of widespread tissue damage.

Chronic cerebellar stimulation in the monkey Electron microscopic and biochemical observations

The effects of chronic electrical stimulation to the surface of cerebellum in the Macaca mulatta monkey were studied with morphologic and biochemical techniques. There was considerable damage and loss of Purkinje cells in all specimens examined, including an area without electrodes, but the greatest changes appeared in tissue beneath the cathode and anode. Despite the damage, normal appearing synapses persisted in the molecular layer of all specimens. Fibrous glial processes were more numerous beneath the cathode. There were abnormalities in gamma-amino butyric acid (GABA) and polyamine concentrations in virtually all specimens, consistent with the morphologic evidence of widespread tissue damage.

Antiviral activity of oxidized polyamines and aldehydes

Abstract Amine oxidase was isolated from lamb serum, purified 50-fold by Chromatographic techniques, and used in the preparation of the aminoaldehydes of spermine and spermidine. Crude spermine aldehyde inactivated a number of animal viruses, but failed to affect virus hemagglutination. Chromatographic fractionation of the aldehyde of spermidine or spermine indicated that fractions containing peak amine aldehyde concentrations produced maximal inactivation of vesicular stomatitis virus. Inactivation of vesicular stomatitis virus by column-purified oxidized spermidine (a monoaldehyde) or spermine (a dialdehyde) was a function of the concentration of aldehydic groups. Viral inactivation produced by the amine aldehydes proceeds in a linear-first-order reaction with respect to time. Studies to determine structure-antiviral activity relationships were conducted with a number of aliphatic and aromatic aldehydes. These results suggest that the aldehyde moiety is the primary determinant of antiviral activity.

Polyamines in normal and cataractous human lenses: Evidence for post-translational modification

The polyamine composition of normal and cataractous human lenses has been studied. Polyamines (putrescine, spermidine and spermine) have been shown to occur in the unbound form in the acid-soluble fraction and in the bound form in the acid-insoluble fraction of the lens. It has also been shown that only putrescine occurs in both conjugated and non-conjugated states in the lens, while putrescine as well as spermidine occur in both forms in the aqueous fluid. Estimation of the polyamine content in cataractous lenses indicates elevated levels of both bound and unbound polyamines in these lenses in comparison to the normal lenses. Evidence is presented which indicates an increase in the level of polyamines covalently bound to protein, in a gamma-glutamyl linkage, in the cataractous lens.

GABA and its relationship to putrescine metabolism in the rat brain and pancreas

The possibility that GABA may have its origin in putrescine was investigated in the rat pancreas, relative to the brain. These studies show that radioactive putrescine is converted to GABA at a similar rate in both the pancreas and brain, but that putrescine accounts for only a small fraction of the GABA found in these organs. Inhibitors of diamine and monoamine oxidases do not significantly change the GABA level in the pancreas. In contrast to the brain, where putrescine is catabolized to GABA via monoamine oxidase, the primary catabolic pathway of putrescine to GABA in the pancreas is via diamine oxidase. In vivo studies show that AOAA inhibits GABA-T activity to the same degree in the pancreas as in the brain, elevating GABA levels more than 2-fold in 4 h. GABA is metabolized more rapidly in the brain than the pancreas. Turnover times of GABA in the pancreas and brain are 1.9 and 1.0 h, respectively. The slower turnover of GABA in the pancreas than in the brain may relate to a neuromodulatory role for GABA, similar to that for neuropeptides. Developmental studies in the postnatal pancreas suggest a role for GABA in the maturation of insulin secretion.

Polyamine biosynthesis and vitamin B6 deficiency Evidence for pyridoxal phosphate as coenzyme for S-adenosylmethionine decarboxylase☆

Abstract Extracts of liver from vitamin B 6 -deficient rats had only 50% of the S -adenosylmethionine decarboxylase activity of extracts of liver from control rats when assayed with no exogenous pyridoxal phosphate. When pyridoxal phosphate was included in the reaction mixture, both extracts exhibited the same activity, indicating that pyridoxal phosphate is the coenzyme for S -adenosylmethionine decarboxylase. There was no similar decreased activity in extracts of brain from vitamin B 6 -deficient rats. The activity of the pyridoxal phosphate-dependent enzyme, ornithine decarboxylase, was increased in extracts of liver from vitamin B 6 -deficient rats: 1.6-fold when assayed with no pyridoxal phosphate and 4-fold when assayed with pyridoxal phosphate. The concentrations of putrescine and spermidine were decreased 50% in liver of vitamin B 6 -deficient animals, but only putrescine was decreased in brain. Putreanine was barely detectable in liver of vitamin B 6 -deficient animals, but was unchanged in brain.