Jack D. Klingman

Acetyl- and pseudocholinesterase activities in sympathetic ganglia of rats.

—The quantitative method of Ellman, Courtney, Andres and Featherstone (1961) was adapted to a differential assay for the determination of acetyl‐ and pseudocholinesterase activities of sympathetic ganglia of rats. The activities of the cholinesterases of superior cervical, stellate and thoracic chain ganglia and of the abdominal ganglionic complexes in apposition to the superior mesenteric and coeliac arteries (superior mesenteric, coeliac and cardiac ganglia) were measured. B.W.284C51 dibromide, 5 × 10−5m, and ethopropazine hydrochloride, 3·15 × 10−5m, were employed to inhibit selectively acetyl‐ and pseudocholinesterases, respectively. Linearity was shown to be maintained with enzyme concentrations corresponding to 0·12‐0·5 mg of ganglion (wet wt.)/incubation. Under the experimental conditions of this assay, the rates of the reaction of ganglionic acetyl‐ and pseudocholinesterases were linear for time periods greater than those employed for calculating the rates of hydrolysis in the homogenates of sympathetic ganglia. Several experimental approaches were used to ascertain the specificity of the inhibitors and of the reaction.

Catecholamines in peripheral tissues of mice and cell counts of sympathetic ganglia after the prenatal and postnatal administration of the nerve growth factor antiserum.

One group of mice was exposed prenatally on the 15th, 16th and 17th day of gestation to the Nerve Growth Factor-antiserum (900 units/g per day), two other groups of mice were, after such prenatal treatment, also treated with 450 or 900 units/g per day postnattly for 6 days and a fourth group was treated postnatally only with 450 units/g per day for 6 days. One-and-a-half to71/2 months following such treatments the catecholamine content of major peripheral tissues was measured and total cell counts were performed on 107 sympathetic ganglia (superior cervical, stellate, superior mesenteric, celiac and cardiac ganglia) from anti-serum-treated and control mice. Prenatal exposure to the antiserum caused a moderate decrease in ganglionic cell counts with a corresponding moderate decrease of norepinephrine levels in submaxillary glands and small intestine; insignificant decreases in renal and uterine norepinephrine concentrations were seen, while the spleen and heart were nearly devoid of norepinephrine. Postnatal antiserum treatment was more effective causing marked reductions of tissue norepinephrine levels and ganglionic cell populations. The combined pre- and postnatal treatments gave the greatest effects. The norepinephrine levels were reduced to consistently negligible quantities in the heart, spleen, submaxillary glands and small intestine and renal and uterine norepinephrine concentrations were also significantly decreased. Only 1·7–8·2 per cent of the cell populations of the various sympathetic ganglia survived the combined pre- and postnatal treatments.

Effects of lithium ions on the rat superior cervical ganglion

Abstract Rat superior cervical ganglia, when stimulated in lithium-containing bathing solution, show decreasing action potentials with increasing lithium concentrations. When 70% of the sodium is replaced by lithium there is an irreversible loss of the action potential. This is not reversed by returning the ganglion to normal bathing solution, with or without stimulation. The decrease in ganglionic transmission is similar when only the pre-ganglionic fibers or the ganglionic body and the post-ganglionic fibers are exposed to lithium-containing bathing solution (compartmentalized cell). The experiments suggest that lithium has two effects; the usual depression of the action potential and the more sensitive action on the synaptical transmission.

Cholinesterases of rat sympathetic ganglia after immunosympathectomy, decentralization and axotomy.

Abstract— Immunosympathectomy was produced in Sprague‐Dawley rats by the subcutaneous injection of 300 units of nerve growth factor (NGF)‐antiserum (1.56 mg of freeze‐dried serum)/g/day for 6 days, the first dose being given 5–8 hr after birth. The immunosympathectomized rats and their control littermates were killed 2½ and 7 months after birth.

The effects of electrical stimulation and ionic alterations on the metabolism of amino acids and proteins in excised superior cervical ganglia of the rat.

Abstract— The effects of supramaximal electrical stimulation on the metabolism of amino acids and proteins in incubated superior cervical ganglia of the rat were studied by the use of a gas‐liquid chromatographic (GLC) assay procedure. Stimulation at 5 Hz for 2 h caused an apparent increase in tissue levels of free amino acids, with alanine, serine, glycine, valine, threonine, isoleucine and aspartate (+ asparagine) most noticeably affected. The amino acid composition (partial) of the TCA‐insoluble proteins of resting and stimulated ganglia was approximately the same after 60 min of incubation, but there was less TCA‐insoluble protein in the stimulated ganglia. The addition of amino acids (at plasma concentrations) to the standard media had no apparent affect on the amino acid composition of this protein fraction. Stimulation for 0, 5 h initially increased the efflux of alanine, valine, proline and ornithine into the incubation media but prolonged stimulation (for 4–0 h) decreased the efflux of alanine, serine, glycine and isoleucine and increased the efflux of lysine into the incubation media. The leakage of amino acids from the ganglia appeared to be a sodium‐dependent process.

Detection, determination, and metabolism in vitro of gangliosides in mammalian sympathetic ganglia.

Abstract— Sympathetic ganglia of the rat and cat were examined for the occurrence and distribution of gangliosides. Each rat superior cervical ganglion contained 0.3 nmol of ganglioside‐sialic acid. Extracts of cat superior cervical and nodose ganglia were chromatographed on silica gel thin‐layer plates. The resulting patterns suggested that similar distributions of multiple forms of gangliosides occur in these two tissues, with the fast‐moving gangliosides predominating. The metabolic activity of gangliosides was also investigated in rat superior cervical ganglia in vitro. Evidence was obtained that 14C from [U‐14C]glucose, [U‐14C]pyruvate, and [U‐14C]glucosamine was incorporated into the gangliosides.

Single-column gas chromatographic separation of nanomolar quantities of amino acids

Abstract Three gas chromatography packings are reported that can be used to separate mixtures of amino acids present in nanomolar quantities. These packings can be used with single column operation without bleed interference. The best separation was achieved with 1.2% PDEAS on Gas Chrom A which had been carefully prepared and conditioned. Preliminary data are presented which indicate that gas chromatography can be used to analyze amino acids present in nanomolar quantities in biological samples.

Incorporation of rdiolabelled sugars into synaptic junctional macromolecules from chick brain

Individual radiolabelled sugars ([1-14C]-L-fucose, [U-14C]-D-galactose, [U-14C]-D-mannose, [U-14C]-D-glucose and [U-14C]-D-glucosamine) were injected intraventricularly into the forebrains of day-old chickens, and the relative incorporation, after 6 hr, into the glycoprotein sugars, aglycosyl protein, glycolipid sugars and aglycosyl lipid assessed. In addition, the relative total and specific activities of the individual glycoprotein sugars was also determined. Exclusive labelling of glycoprotein sugars (and of the fucose within these oligosaccharides) using fucose as precursor was confirmed, while optimal labelling of glycolipid sugars was achieved using the precursor mannose. Galactose, mannose, glucose and glucosamine were converted to varying degrees to other sugars and non-sugar precursors prior to incorporation. Glucosamine gave rise to the most even distribution of radioactivity among the glycoprotein sugar residues. The highest relative specific activities of the glycoprotein oligosaccharides were achieved using fucose and glucosamine as precursors.

Monoamine oxidase activity in peripheral organs and adrenergic tissues of the rat.

Abstract Monoamine oxidase activity in vitro was studied in the heart, kidneys, submaxillary glands, and superior cervical ganglia of control rats by the method of Lovenberg and co-workers ( J. Pharmacol . 1962). Comparison of monoamine oxidase activity in the absence and presence of exogenous aldehyde dehydrogenase revealed nearly saturating amounts of aldehyde dehydrogenase in the liver, submaxillary glands, lungs, spleen, and stomach (fundus, body, pyloric antrum). In the absence of exogenous aldehyde dehydrogenase, the uterus, ventricles, right atrium, left atrium, and proximal and distal small intestine showed activities of 24, 46, 58, 49, 65, and 68 per cent activity respectively. The activity in the superior cervical ganglia, stellate ganglia, thoracic chains, and retinas was 8.5, 11.0, 27, and 38 per cent. In the presence of exogenous aldehyde dehydrogenase, the greatest monoamine oxidase activity, based on the protein content, was noted in the superior cervical ganglia. On the wet weight basis, ganglionic monoamine oxidase activity was greater than that of all other tissues investigated except the liver. In the absence of exogenous aldehyde dehydrogenase, the reaction velocity of monoamine oxidase in ventricles, atria and pooled adrenergic tissue was linear for 30 min at the optimum substrate concentration. In the presence of exogenous aldehyde dehydrogenase, the monoamine oxidase reaction rates of the ventricles, atria, and adrenergic tissue were linear for up to 40 min at optimum substrate concentrations. Substrate inhibition became apparent at the highest substrate concentration used (11.67 mole/ml), and unsaturation of the enzyme was noted at the lowest substrate concentration (0.233 mole/ml). In homogenates of the ventricles, atria, and pooled adrenergic tissue, monoamine oxidase activity was inhibited by relatively small increases in enzyme concentration (tissue homogenate). This inhibition was not overcome by a tenfold increase in substrate concentration nor by the doubling of NAD and exogenous aldehyde dehydrogenase. Centrifugation at 50,000 g for 25 min localized the inhibitory component(s) in the pellet. The supernatant was devoid of monoamine oxidase activity.

Synaptic junctional glycoconjugates from chick brain. Glycoprotein identification and carbohydrate composition.

Forebrains from day-old chicks were homogenized and fractionated by differential sedimentation and density gradient centrifugation to yield subcellular fractions. The synaptosomal plasma membrane fraction was further treated with Triton X-100 to yield subsynaptic membrane fractions including synaptic junctions. Glycoproteins from these subsynaptic membrane fractions were identified after separation by SDS-polyacrylamide gel electrophoresis by incubating the gel slabs with radioiodinated concanavalin A. Two lectin-binding proteins were discerned in the synaptic junction fraction while none were observed in the Triton-soluble portion of the synaptic plasma membrane. The carbohydrate content of the glycoproteins from each subcellular fraction was quantitated after methanolysis and derivatization aso-methyl-trifluoroacetyl analogs by gas-liquid chromatography. The lowest concentration of glycoprotein sugars was found in the synaptic junction, mitochondrial, and soluble fractions while the greatest concentration was found in the myelin, light-synaptic plasma membrane, and the Triton-soluble portion of the synaptic plasma membrane. Of the subcellular fractions, the synaptic junction contained the highest porportion of mannose and lowest proportion of sialic acid. Moreover, this fractions content of galactose andN-acetylglucosamine, relative to mannose was the lowest while its content of fucose was low. The oligosaccharide chains extending into the synaptic cleft therefore are predominantly of the “neutral, mannose-rich” type and are attached to a limited number of high-molecular-weight glycoproteins.