Gertrude M. Tyce

Efflux of 5‐hydroxytryptamine and noradrenaline into spinal cord superfusates during stimulation of the rat medulla.

High pressure liquid chromatography with electrochemical detection was used to quantify the efflux, in the same sample, of endogenous 5‐hydroxytryptamine (5‐HT), noradrenaline (NA), and 5‐hydroxyindoleacetic acid (5‐HIAA) into superfusates of the rat spinal cord in vivo. The efflux of these three agents was measured prior to, and during, electrical stimulation of the nucleus raphe magnus (n.r.m.) and nucleus reticularis paragigantocellularis (n.r.p.g.), two medullary nuclei implicated in antinociception. In untreated rats, basal efflux of 5‐HT and NA was 0.21 and 0.12 ng/ml of superfusate respectively; the basal efflux of 5‐HIAA was 18.17 ng/ml. Stimulation of the n.r.m. and n.r.p.g. in these animals increased the efflux of 5‐HT and 5‐HIAA, but did not alter the efflux of NA. 60 min after administration of fluoxetine (10 mg/kg, I.P.), a 5‐HT uptake inhibitor, basal efflux of 5‐HT and NA was unaltered, but the basal efflux of 5‐HIAA was decreased. In these rats, stimulation of the n.r.m. and n.r.p.g. increased the efflux of 5‐HT and of NA. The efflux of 5‐HIAA was not altered. In rats pre‐treated with both fluoxetine and desipramine (10 mg/kg, I.P.), the basal efflux of NA was increased while that of 5‐HIAA was decreased; the basal efflux of 5‐HT was not affected. The efflux of NA, but not of 5‐HT, was increased in these animals during stimulation of the n.r.m. and n.r.p.g. The efflux of 5‐HIAA was not changed by stimulation. Addition of fluoxetine alone or with desipramine to the superfusate in high concentrations greatly increased basal efflux of 5‐HT. Failure of stimulation of the ventromedial medulla to increase the efflux of 5‐HT in these animals may be related to feed‐back inhibition of release by the high concentration of 5‐HT initially present in the superfusate. These results indicate that electrical stimulation of the n.r.m. and n.r.p.g. increases the efflux of endogenous 5‐HT and NA from the spinal cord. These stimulation sites are coincident with brain‐stem sites at which stimulation produces antinociception by activation of spinal serotonergic and noradrenergic receptors. Thus, the ability of stimulation at these sites to evoke the spinal release of the probable neurotransmitters further supports the hypothesis that the antinociceptive effect is mediated by activation of serotonergic and noradrenergic neurones projecting to the spinal cord.

Decreased adrenal medullary catecholamines in adrenal transplanted parkinsonian patients compared to nephrectomy patients.

Adrenal medullary catecholamines were measured in tissue samples from eight patients who underwent autologous transplantation of the adrenal medulla to the caudate nucleus as a treatment for Parkinsons disease. These adrenal catecholamine levels were compared to a group of patients of similar age who underwent unilateral nephrectomy for renal cell carcinoma. The levels of each catecholamine, expressed as nanomoles per milligram wet weight tissue, were significantly lower (P less than or equal to 0.005) in the parkinsonian patients than in the nephrectomy patients. These observations support data reported previously from autopsy specimens and suggest that the adrenal medullae of parkinsonian patients may be a compromised source of dopamine-producing tissue; this may limit its effectiveness in eliciting maximum clinical improvement following transplantation.

Spinal cord monoamines modulate the antinociceptive effects of vaginal stimulation in rats

Abstract Perispinal administration (into the lumbar intrathecal space) of phentolamine (40 &mgr;g), an &agr;‐adrenergic receptor blocking agent, reduced the analgesic effect of vaginal stimulation by 39.7% (measured by tail flick latency) and 57.1% (measured by vocalization threshold) as compared to controls. Perispinal administration of methysergide (10 &mgr;g), a serotoninergic receptor blocking agent, reduced the analgesic effect of vaginal stimulation by 48.5% (measured by vocalization threshold), although it did not significantly affect the tail flick measure. In a separate experiment, vaginal stimulation activated the release of norepinephrine and serotonin into a superfusate of the spinal cord. During vaginal stimulation, levels of norepinephrine and serotonin increased about 2‐fold above resting levels. These findings indicate that vaginal stimulation releases norepinephrine and serotonin into the spinal cord, thereby exerting an analgesic effect.

3-Methoxy-4-hydroxyphenylalanine (3-O-methyldopa) in plasma during oral L-dopa therapy of patients with Parkinson's disease

Abstract A new fluorometric method based on the hydroxyindole reaction was developed for determination of 3- O -methyldopa in plasma of patients being treated with L -dopa. Dopa and homovanillic acid also were measured fluorometrically in the same plasma samples. During the 5-h period after a first dose of L -dopa was given to patients with Parkinsons disease, plasma 3- O -methyldopa levels increased, but more slowly and to a lesser degree than either dopa or homovanillic acid. After prolonged L -dopa therapy, 3- O -methyldopa accumulated in plasma, usually reaching levels higher than those of dopa. During the first 4 h after the morning dose of dopa in patients on long-term therapy, 3- O -methyldopa levels in plasma decreased slightly, while dopa and homovanillic acid increased as in patients newly treated.

5-hydroxyindole metabolism in the brain after hepatectomy.

Abstract The concentrations of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) increased in the brain of dogs and rats after complete hepatectomy, the increases of 5-HIAA being greater than those of 5-HT. The increase in concentration of both 5-hydroxyindoles was much smaller than that of the precursor, tryptophan (Try). The demonstration of radioactive 5-HT and 5-HIAA in the brain of normal and hepatectomized rats after the intradiencephalic injection of l -Try-3- 14 C indicated that hydroxylation of Try occurred in brain in vivo . However, the intravenous injection of Try (33 mg/kg) resulted in definite increases in the concentration of 5-hydroxyindoles in the brain of normal rats and only marginal increases in the brain of hepatectomized rats. After intravenous injection of d , l -5-hydroxytryptophan, conversion to 5-HT was essentially similar in normal dogs and in dogs in coma after complete hepatectomy.

Glucose and amino acid metabolism in rat brain during sustained hypoglycemia.

The metabolism of glucose in brains during sustained hypoglycemia was studied. [U-14C]Glucose (20 μCi) was injected into control rats, and into rats at 2.5 hr after a bolus injection of 2 units of insulin followed by a continuous infusion of 0.2 units/100 g rat/hr. This regimen of insulin injection was found to result in steady-state plasma glucose levels between 2.5 and 3.5 μmol per ml. In the brains of control rats carbon was transferred rapidly from glucose to glutamate, glutamine, γ-aminobutyric acid and aspartate and this carbon was retained in the amino acids for at least 60 min. In the brains of hypoglycemic rats, the conversion of carbon from glucose to amino acids was increased in the first 15 min after injection. After 15 min, the specific activity of the amino acids decreased in insulin-treated rats but not in the controls. The concentrations of alanine, glutamate, and γ-aminobutyric acid decreased, and the concentration of aspartate increased, in the brains of the hypoglycemic rats. The concentration of pyridoxal-5′-phosphate, a cofactor in many of the reactions whereby these amino acids are formed from tricarboxylic acid cycle intermediates, was less in the insulin-treated rats than in the controls. These data provide evidence that glutamate, glutamine, aspartate, and GABA can serve as energy sources in brain during insulin-induced hypoglycemia.

Free and conjugated dopamine in human ventricular fluid

Free dopamine and an acid hydrolyzable conjugate of dopamine were measured in human ventricular fluid specimens with a radioenzymatic assay and by high performance liquid chromatography (HPLC) with electrochemical detection. Only trace amounts of free norepinephrine and dopamine were detected in ventricular fluid from patients with movement disorders. When the ventricular fluid was hydrolyzed by heating in HClO4 by lyophilization in dilute HClO4, however, a substantial amount of free dopamine was released. Values for free plus conjugated dopamine in ventricular fluid from patients who had never taken L-DOPA ranged from 139 to 340 pg/ml when determined by HPLC and from 223 to 428 pg/ml when measured radioenzymatically. The correlation coefficient for values obtained by the two methods in the same sample of CSF was 0.94 (P less than 0.001). Patients who had been treated with L-DOPA had higher levels of conjugated dopamine in their ventricular CSF which correlated inversely with the time between the last dose of L-DOPA and withdrawal of the ventricular fluid. Additionally, one patient with acute cerebral trauma had elevated levels of free norepinephrine and both free and conjugated dopamine in his ventricular fluid. Conjugation may be an important inactivation pathway for released dopamine in man.

Current Experience in the Diagnosis of Pheochromocytoma

The pharmacological and biochemical tests available for the diagnosis of pheochromocytoma were evaluated among 28 patients with proved tumors and 148 other patients in whom the diagnosis was suspected. Of the biochemical tests on urine, determinations of total metanephrines (MN) and of vanilmandelic acid (VMA) were much less subject to false-positive results than was determination of catecholamines (CA). Since urinary MN values changed the most in tumor patients, MN determination is preferred as the current screening test. Of the pharmacological tests, the histamine test continues to be the most valuable, especially when combined with determination of plasma CA concentration. The phentolamine (Regitine) test has not proved to be of any distinct value as a primary diagnostic aid. Any of the chemical or pharmacological tests occasionally may give a false-positive result. The urinary output of excessive amounts of homovanillic acid (HVA) was an indication of the presence of pheochromocytoma with some features histologically similar to other neural crest tumors but was not an indication of malignancy. Reliable diagnosis of pheochromocytoma still must be based on multiple studies in an appropriate clinical setting.

The secretion of catecholamines, chromogranin A and neuropeptide Y from the adrenal medulla of the cat via the adrenolumbar vein and thoracic duct: Different anatomic routes based on size

Secretion of the adrenal medulla was stimulated in nine cats by insulin-induced hypoglycemia. Levels of catecholamines (mol. wt 153-183), neuropeptide Y (mol. wt 4254) and chromogranin A (mol. wt 48,000) were measured in concurrently collected samples of adrenolumbar venous blood and thoracic duct lymph for up to 4 h following insulin administration. Insulin-induced hypoglycemia elicited an increase in the secretion of catecholamines, which reached peak levels in the adrenolumbar venous plasma at 1.5-2 h and in the lymph at 2.5 h. Although catecholamines were the most numerous measured molecules in the lymph, levels of norepinephrine and epinephrine were 75-250-fold less than those found in the adrenolumbar venous plasma. Neuropeptide Y in the adrenolumbar venous plasma reached peak levels between 1 and 1.5 h; at this time approximately 20% of the peak venous amount was detected in the lymph. Chromogranin A was found in approximately equal amounts in both plasma and lymph; the peak level in the plasma occurred at 1.5-2 h, while that in the lymph was reached at 2-3 h. We suggest that the size of a molecule influences the route it takes following exocytosis from the chromaffin vesicle. Smaller molecules such as catecholamines may pass directly into the circulation, while larger molecules such as chromogranin A may be temporarily sequestered in the interstitial space before passing into the lymph, and hence into the circulation.

Concurrent separation of catecholamines, dihydroxyphenylglycol, vasoactive intestinal peptide, and neuropeptide Y in superfusate and tissue extract

A method is described for separation and quantification of 3,4-dihydroxyphenylglycol (DO-PEG), norepinephrine (NE), dopamine (DA), vasoactive intestinal peptide (VIP), and neuropeptide Y (NPY) from single samples of tissue homogenate and from superfusate from in vitro dog blood vessel preparations using cartridges containing 0.4 g of octadecylsilane (Sep-Pak C-18). Samples were passed through the cartridge at pH 7.4. A step-gradient system was used to first selectively desorb the catechols (DOPEG, NE, DA) with a moderately polar eluent; subsequently VIP and NPY were eluted with 2.5 ml of a mixture of 1% trifluoroacetic acid, 80% acetonitrile. Five Sep-Pak catechol eluents were tested. Catechols were quantified by HPLC with electrochemical detection and peptides by radioimmunoassay. An HPLC solvent system is described which is particularly useful for chromatography of the more hydrophilic catechols DOPEG, 3,4-dihydroxymandelic acid, and 3,4-dihydroxyphenylalanine concurrently with catecholamines. For superfusion studies, sample cleanup time was reduced to about 4 min per sample by attachment of the cartridges directly to the bottom of the superfusion chamber. Superfusate was subsequently pulled through the cartridges immediately after they were passed over the tissue. Batches of 12 high-speed tissue supernates were processed through the method in about 30 min. The method was used to analyze DOPEG, NE, DA, VIP, and NPY in various rat and dog tissues. The values obtained were similar to values obtained previously by other methods. Because the catechols and peptides are separated from a single sample, the method has several advantages over those described previously; e.g., it is rapid, simple, and more sensitive.