Jerome A. Levin

Paper chromatographic assay of [3H]norepinephrine and its five major metabolites

Abstract The technique described here offers several advantages over the currently available assays for [3H]norepinephrine and its metabolites. The technique is simpler and has fewer sources of potential error than most currently available techniques. It provides a complete separation of norepinephrine from its five major metabolites with virtually no cross-contamination between the fractions. It is unnecessary to correct for recovery, elution from the chromatograms, or efficiency of counting the chromatogram segments. Experiments reported here demonstrate that all five major metabolites of norepinephrine are formed by rabbit aorta. Most of the neutral metabolites and normetanephrine are found in the Krebs solution used for incubating the tissue, whereas most of the acid metabolites are found in the tissue.

Separation of norepinephrine and its five major metabolites by paper chromatography

Abstract Over one-hundred solvent systems were tested on Whatman No. 1 paper and over fifty solvent systems were tested on silica gel loaded paper, cellulose phosphate paper, and carboxymethyl cellulose paper to find a method for separating norepinephrine and its five major metabolites. Although no single solvent system adequately separated all six compounds on any of the papers used, a number of solvents did separate the compounds into chemically related groups. Complete separation of all six compounds was obtained by chromatography in two different solvent systems on cellulose phosphate paper. n -Butanol—95% ethanol—water (I:I:I) separated norepinephrine from normetanephrine with virtually no interference from the deaminated metabolites. n -Butanol—pyridine—water (14:3:3) retained the amines at the origin and provided complete separation of the deaminated metabolites along the remainder of the paper. Very small amounts of sodium chloride increased the mobility of the amines on the cation-exchange papers.

Effect of Inhibitors of Neuronal and Extraneuronal Uptake on the Accumulation and Metabolism of 3H-l-Norepinephrine in Rabbit Aorta

In the isolated adventitia of rabbit aorta, blockade of neuronal uptake decreased the accumulation and deamination of 3H-l-norepinephrine (3H-l-NE) whereas blockade of extraneuronal uptake decreased O-methylation. Thus most of the O-methylation occurs in the extraneuronal elements. In the isolated media, blockade of extraneuronal uptake decreased metabolism but not accumulation of 3H-l-NE. If NE metabolism was prevented by pretreatment with pargyline and U-0521, 3H-l-NE accumulation in the isolated media increased markedly and now inhibitors of extraneuronal uptake could decrease this accumulation of 3H-l-NE by the isolated media. Thus when MAO and COMT are intact, NE does not accumulate in the extraneuronal compartment containing these enzymes, probably because it is metabolized as fast as it enters the compartment. Since the level of 3H-l-NE in the isolated media exceeded the sorbitol space when MAO and COMT are intact, some 3H-l-NE must have accumulated in a compartment which does not contain these enzymes.

Two Mechanisms of 3H-Catecholamine Accumulation in Rabbit Aorta Media Differentiated by Sensitivity to Temperature and Corticosterone

To characterize the extraneuronal accumulation of catecholamines (CA) in the media of rabbit aorta pieces of isolated media were incubated with 3H-CA (norepinephrine, isoproterenol or normetanephrine) plus 14C-sorbitol (to estimate the extracellular space) for varying periods at 37 or 0 degree C. The 3H-CA accumulation was 1.3 ml/g at 0 degree C and 2.2 ml/g at 37 degrees C. The sorbitol space was 0.6 ml/g at both temperatures. CA accumulation at 0 and 37 degrees C is significantly different from each other and from sorbitol accumulation. Corticosterone and phenoxybenzamine inhibit the temperature-sensitive component of CA accumulation. Accumulation of norepinephrine at 0 degree C is unaffected by corticosterone, phenoxybenzamine or oxytetracycline. The initial rate of NE accumulation at 37 degrees C, from steady state accumulation at 0 degree C, and the initial rate of accumulation at 0 degree C were linear functions of NE concentration between 10(-7) and 10(-2) M. Based on differences in sensitivity to corticosterone and temperature, we conclude that CA accumulation at 0 degree C is different from the accumulation at 37 degrees C.

EXTRANEURONAL UPTAKE AND METABOLISM OF 3H-l-NORADRENALINE IN RABBIT AORTA

Publisher Summary This chapter discusses the extraneuronal uptake and metabolism of 3H-l-noradrenaline in rabbit aorta. The major problem encountered in studying the extraneuronal uptake of noradrenaline is that this process is masked by the more prominent uptake and storage of NA by adrenergic nerve terminals. The effects produced by iproniazid were qualitatively similar to the effects of pargyline. In the method described in the chapter, the tissues were pretreated with 3 × 10−4 M to 1 × 10−2 M iproniazid for 25 min and then washed for pargyline. Iproniazid was 30 to 100 times less potent than pargyline. The highest concentration of iproniazid increased the amount of NM found after incubation of isolated media with 3H-l-NA. This may reflect merely a decrease in the deamination of NM rather than an actual increase in O-methylation of NA. The harmaline was left in contact with the tissue during incubation with 3H-l-NA. The most striking effect of harmaline was the decrease in the formation of all five metabolites of 3H-l-NA. It is found that because harmaline also increased the tissue accumulation of 3H-l-NA these effects probably result from inhibition of both MAO and COMT rather than from inhibition of extraneuronal uptake. Harmaline was a slightly more potent MAO inhibitor than pargyline.