Putrescine, a source of gamma-aminobutyric acid in the adrenal gland of the rat.

Putrescine is the major source of gamma-aminobutyric acid (GABA) in the rat adrenal gland. Diamine oxidase, and not monoamine oxidase, is essential for GABA formation from putrescine in the adrenal gland. Aminoguanidine, a diamine oxidase inhibitor, decreases the GABA concentration in the adrenal gland by more than 70% after 4 h, and almost to zero in 24 h. Studies using [14C]putrescine confirm that [14C]GABA is the major metabolite of putrescine in the adrenal gland. Inhibition of GABA transaminase by amino-oxyacetic acid does not change the GABA concentration in the adrenal gland, as compared with the brain, where the GABA concentration rises. With aminoguanidine, the turnover time of GABA originating from putrescine in the adrenal gland is 5.6 h, reflecting a slower rate of GABA metabolism compared with the brain. Since GABA in the adrenal gland is almost exclusively derived from putrescine, the role of GABA may relate to the role of putrescine as a growth factor and regulator of cell metabolism.

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.

Cerebrospinal fluid polyamines: biochemical markers of malignant childhood brain tumors.

The clinical value of cerebrospinal fluid (CSF) polyamine determinations in childhood medulloblastoma has been suggested. We performed 72 CSF polyamine determinations in 35 children with primary brain tumors. Spermine values were normal and spermidine values were inconsistently elevated. CSF putrescine values, however, were consistently elevated in patients with histologically malignant brain tumors: medulloblastoma, ependymoma, pineal germ cell tumors, primitive neuroectodermal tumors, and brainstem gliomas. Children with supratentorial astrocytomas had normal CSF polyamine values. CSF putrescine values were closely correlated with clinical state, with the highest concentrations identified in patients with widely disseminated recurrent disease. We found CSF putrescine to be a sensitive indicator of active disease in childhood malignant brain tumors. Further investigation is warranted into the predictive value of CSF polyamines in determining tumor relapse before clinical or other diagnostic studies reveal recurrent disease.

Alpha-bungarotoxin binding to the myotome and choline acetyltransferase activity in the rabbit embryo.

We have previously found incomplete sarcomeres and acetylcholinesterase activity in the myoblasts of the myotome of the rabbit at day 13 of gestation. We now report that an acetylcholine (ACh)-synthesizing enzyme and the nicotinic receptor are present at this stage as well. A study of the myotome using [125I]alpha-bungarotoxin shows that the mononucleated myoblasts have alpha-bungarotoxin binding sites before they migrate away to form multinucleated myotubes. Choline acetylcholinesterase activity and/or a different ACh-synthesizing enzyme are found at early stages of development, even before the spinal nerve has formed. An ACh-synthesizing enzyme is present in the notochord, a neural tube-dorsal root ganglion preparation, as well as in rows of myotomes separated from the latter preparation. Assays of isolated myotomes with very little adherent mesenchyme indicate that the enzyme is located either within the myotome or in its immediate vicinity. Cholinergic components, therefore, are associated with the mononucleated myoblasts of the myotome before they fuse to form myotubes and before they receive their permanent innervation.

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.

Metabolism and axonal transport of polyamines in a single identified neuron of Aplysia californica.

The metabolism of polyamines was investigated by injecting purified [3H]putrescine directly into the soma of the giant neuron R2 of Aplysia. Injected putrescine was rapidly metabolized to spermidine, spermine, and several catabolites, including GABA and monoacetylputrescine. Identification of these products was by comparison with the authentic compound using ion exchange chromatography. When R2 was injected with amounts of [3H]putrescine determined so that the intracellular content of labeled precursor was less than 6 X 10(-6) M, metabolism was rapid and occurred via pathways similar to those in mammalian tissues. At concentrations of labeled precursor greater than 2 X 10(-4) M, relatively little putrescine was converted to product. By 4 h after injection, putrescine and its labeled products appeared in R2's axon, where additional metabolism occurred. These results indicated that the enzymes involved in polyamine interconversion are not restricted to R2's cell body, and this suggestion was corroborated by finding ornithine decarboxylase and S-adenosylmethionine decarboxylase activities in Aplysia nerves. The distribution of the polyamines along R2's axon was compared with that of 3H-glycoproteins, with the finding that while the acid-soluble polyamines move by diffusion, labeled polyamines associated with protein are rapidly transported.

Post-translational modification of neuronal proteins: evidence for transglutaminase activity in R2, the giant cholinergic neuron of Aplysia.

[3H]Putrescine injected into the cell body of the giant neuron R2 of Aplysia was readily converted to gamma-aminobutyric acid, acetylputrescine, spermidine, and spermine. In addition, labeled putrescine and spermidine were found covalently linked to protein through the action of an intracellular transglutaminase. This was shown by exhaustively treating the acid-insoluble fraction from injected cells with Pronase, aminopeptidase M, and carboxypeptidases A and B. High-performance liquid chromatography of the digest revealed labeled gamma-glutamylputrescine and gamma-glutamylspermidine, the products expected from the transglutaminase-catalyzed post-translational modification of intracellular proteins. In vitro assays of Aplysia nervous tissue showed the presence of transglutaminase as well as gamma-glutaminyl cyclotransferase, an enzyme that cleaves the gamma-glutamylpolyamine bond. Incorporation of polyamine into proteins in R2 is a specific process because only a few 3H-labeled polypeptides were found after injection.

Polyamines and membrane proteins in sickle cell disease.

The polyamines, putrescine, spermidine, and spermine have been implicated in cellular growth and membrane stabilization. We have shown that RBC stroma and lysate polyamines are significantly elevated in sickle cell disease (SS and SC) relative to controls (AA). The crude spectrin fraction of the red cell stroma has associated with it a large fraction of the total stroma polyamines, suggesting that the polyamines in the stroma are preferentially bound to spectrin. The high levels of polyamines associated with the spectrin of young and ISC's relative to normal may contribute to an alteration of the physical-chemical properties of the sickle RBC membrane. Spermine levels were high in the SS lysates but not detectable in SC or AA lysates. Levels in SC patients were intermediate. In preliminary experiments using washed red cells obtained from SS and AA subjects, there was extensive incorporation of 14C putrescine into red cell protein. Labelled N-(gamma-glutamyl) putrescine was isolated, consistent with covalent binding of the polyamines to protein.

Polyamines and globin binding in sickle cell disease.

Erythrocytes obtained from patients with sickle cell anemia contain five to ten times more spermine than controls. The preferential binding of beta s globin to red cell stroma may be mediated by spermine and may account in part for the membrane abnormalities found in sickle cell disorders.

Long-term surface stimulation of the cerebellum in the monkey. II. Electron microscopic and biochemical observations.

Phase and electron microscopy were used to study the effects of chronic intermittent or continuous stimulation applied to the surface of the cerebellum with bipolar platinum electrodes in monkeys. Damage under the anodes consisted of swelling of neuropil, variable loss of Purkinje cells, and a variable increase in fibrous glial processes. Under the cathode there was marked loss of Purkinje cells, thinning of the molecular layer, moderate swelling, occasional phagocytes with lipofuscin granules, and often an increase of fibrous glial processes. Despite the damage, normal appearing synaptic terminals remained in all layers under both anode and cathode, though sparser in fibrotic areas Gamma-aminobutyric acid (GABA) concentrations were decreased beneath the cathodes, reflecting the loss of Purkinje cells and other GABA-containing cells, and spermidine concentrations were increased in areas with increased fibrous glial processes.

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 alpha-bungarotoxin binding sites and chemosensitivity in developing and denervated muscle.

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.

Electron-microscopic, cytochemical and biochemical studies of acetylcholinesterase and butyrylcholinesterase activity in muscle of dystrophic mice.

We have studied extrajunctional muscle of control and dystrophic mice by electron microscopic-cytochemistry and radiometric assay. We have found both a soluble and particulate AChE activity, which is similar proportionally in control and dystrophic muscle. The particulate AChE activity is probably due to the enzyme localized in the sarcotubular system. These sites are more numerous in muscle adjacent to the motor end-plant than in distally located extrajunctional muscle, and are increased markedly in the dystrophic mouse. Myoblasts and small muscle fibers in the dystrophic mouse also have AChE activity in the reticulum similar to fetal muscle. The soluble AChE activity identified radiometrically may represent those sites exhibiting random cytochemical end-product, such as some muscle nuclei, satellite cells, myogenic mononuclear cells in the connective tissue, and degenerating axonal boutons no longer associated with junctional folds of muscle. Enzyme activity is present in degenerating fibers, but it is randomly dispersed in the sarcoplasm rather than membrane-bound. AChE activity has not been found in debris of completely necrotic muscle. BuChE activity is higher and the number of BuChE-active sites in the sarcotubular system adjacent to the motor end-plates is greater in dystrophic muscle than in control muscle.

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.