Gerda I. 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.

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.

In utero immunosympathectomy of mice

Abstract The antibody for mouse submaxillary gland nerve growth-promoting protein crosses the placenta when injected into pregnant mice and produces a partial immunosympathectomy in the fetus. A dose schedule of 300 units/g twice a day during the 11th to 16th or 12th to 17th day of gestation resulted in significant decreases in the tissue norepinephrine content of the spleen, submaxillary glands and heart (15.5, 25 and 39 per cent, respectively, of control values). Nonrepinephrine levels in other tissues (lungs, gastro-intestinal tract, kidneys and uterus) were not significantly decreased. Injection of the antiserum once a day (300 units/g) during the 13th to 18th day of gestation resulted in a less pronounced reduction in norepinephrine content of the spleen and submaxillary glands. Exposure of the fetus to the antibody during the 7th to 12th day of gestation produced less marked decreases of norepinephrine levels in the spleen and the heart, but no siignificant changes in the norepinephrine content of the submaxillary glands. The latter results could indicate that during the earlier stages of development the postganglionic sympathetic neurones are too immature to be affected, that regeneration or repair takes place or that the antibody does not readily cross the placenta. Cell counts of superior cervical ganglia showed that prenatal antiserum treatment decreased the cell population by approximately 50 per cent. Catecholamine levels in the brain and the adrenal medulla were not affected by the prenatal treatment with the antiserum.

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.

Monoamine oxidase activity of peripheral organs and sympathetic ganglia of the rat after immunosympathectomy

Abstract Treatment of litter-mate rats with the antiserum to the mouse submaxillary gland nerve growth factor of Levi-Montalcini and Cohen resulted in significant decreases in monoamine oxidase activity of the superior cervical, stellate, and thoracic chain ganglia, the spleen, submaxillary glands, kidneys, and liver. The monoamine oxidase activity of the retinas, pineal gland, pituitary gland, lungs, atria, and uterus showed small but statistically insignificant decreases. The monoamine oxidase activity of the ventricles, various portions of the stomach, and proximal and distal small intestine was not affected by treatment with the antiserum. It is postulated that a relation can be shown between sympathetic innervation and monoamine oxidase activity. This can be shown only in those tissues in which the monoamine oxidase associated with sympathetic function constitutes an appreciable portion of the total monoamine oxidase activity. In most of the organs and tissues studied, only a small portion of the monoamine oxidase activity is associated with the nerve endings of the sympathetic postganglionic fibers, whereas the larger portion of the total activity is associated with nonadrenergic sites. Monoamine oxidase activity was measured by the method of Lovenberg and co-workers ( J. Pharmac. exp. Ther. , 1962), with slight modifications found to give reproducible, quantitative results (Klingman and Klingman, Biochem. Pharmac. , 1966).

5-hydroxytryptamine levels in peripheral organs of immunosympathectomized rats

Abstract Littermate Sprague-Dawley rats were immunosympathectomized by the daily injection of Nerve Growth Factor (NGF)-antiserum for 6 days after birth. The 5-hydroxytryptamine concentrations of peripheral tissues, spinal cord and brain stem (medulla, pons, mesencephalon and diencephalon) were measured spectrophotofluorometrically by the method of Bogdanski etal. ‡ In immunosympathectomized rats the 5-hydroxytryptamine levels of the submaxillary glands, hearts and cecum were significantly increased. Statistically nonsignificant elevations were also noted in the lungs, upper intestinal tract, liver and uterus. The submaxillary glands of immunosympathectomized rats were significantly smaller than those of control littermates. The administration of JB-516 (Catron, β-phenylisopropylhydrazine) or 5-hydroxytryptophan did not present evidence to explain the elevated 5-hydroxytryptamine levels in some of the peripheral tissues from immunosympathectomized rats. It is not known whether the rise of the 5-hydroxytryptamine levels in some peripheral tissues from immunosympathectomized rats was a direct result of the NGF-antiserum administration or whether it was due to the fact that these tissues are largely devoid of sympathetic innervation. The most marked and consistent elevation of 5-hydroxytryptamine occurred in those tissues (submaxillary glands and heart) which are nearly completely sympathectomized by the NGF-antiserum administration.

Amine levels, monoamine oxidase and dopa-decarboxylase activities in the gastro-intestinal tract of the rat

Abstract The concentrations of norepinephrine and 5-hydroxytryptamine were found to be highest in the duodenum and proximal jejunum with a gradual decline toward the distal portions of the small intestine. The norepinephrine levels were lower in the pyloric antrum than in the duodenum-proximal jejunum area, whereas 5-hydroxytryptamine levels were similar. Although regional differences in monoamine oxidase and dopa-decarboxylase activities existed in the gastro-intestinal tract of the rat, these regional differences were not correlated with the concentrations of the amines.

Effect of sympathetic denervation on the 5-hydroxytryptamine levels of the submaxillary glands of rats☆

Abstract Sympathetic denervation of submaxillary glands of rats by means of superior cervical ganglionectomy resulted in significant increases of the 5-hydroxytryptamine concentration and total 5-hydroxytryptamine content. Sympathetic denervation reduced significantly the weight of the glands but this decrease was less marked than the previously observed reduction in gland weight following immunosympathectomy. These observations indicated that absence of sympathetic innervation may be responsible for the previously observed increase of the 5-hydroxytryptamine levels in a number of peripheral tissues of rats following immunosympathectomy.

[3H]Spiperone binding in the rat striatum during the development of physical dependence on phencyclidine and after withdrawal.

Binding of [3H]spiperone to dopamine D2 receptors was measured in the striatum of male rats that were infused with phencyclidine (PCP, 45 mg/kg per day) or vehicle (saline) via an intrajugular cannula for 1, 3.5, or 7 days. The 7-day PCP infusion, which was shown previously to induce physical dependence, produced a significant 30% decrease in receptor density (Bmax). Two days after termination of the 7-day PCP infusion, Bmax values were no longer significantly lower than those of saline-infused controls. The acute administration of PCP (20 mg/kg, i.p.) did not alter receptor density 45 min later.