Improved isolation of small noradrenergic vesicles from rat seminal ducts following castration. A density gradient centrifugation and morphological study

Abstract

Noradrenaline storage vesicles from nerve endings have been prepared by differential and density gradient centrifugation. Seminal ducts from castrated rats have been used as a source since fluorescence micrographs and electron microscopy demonstrated an increased concentration of sympathetic nerve endings in this tissue; probably as a result of muscle atrophy. The fractions obtained were analyzed biochemically and partly by electron microscopy. By centrifugation techniques, a density gradient fraction (at the density of 0.4–0.45 m sucrose) was obtained in which noradrenaline was enriched about 10-fold from the homogenate. By using organs from castrated rats a further 4-fold increase was obtained in the noradrenaline/protein ratio of the density gradient fraction. This gradient fraction was studied by electron microscopy, after incubation with 5-hydroxydopamine as an exogenous marker, and was found to contain a high proportion of granular vesicle profiles. More than ⅓ of all vesicles with a diameter below about 1000A˚had an electron dense core. The vesicle preparation contained low activities of mitochondrial (cytochrome oxidase) and lysosomal (acid phosphatase) marker enzymes, while microsomes and plasma membrane fragments as indicated by NADH-cytochrome c reductase and adenosine triphosphatase had only been partially removed and probably constituted the main contaminants. The distribution of dopamine β-hydroxylase activity suggested that the small, light dense cored vesicles are deficient of this enzyme compared with the large, heavy dense cored vesicles, assumed to occur at the bottom of the gradient. The noradrenaline/adenosine 5'-triphosphate molar ratio was found to be 5.9 in the main noradrenaline fraction, but this fraction was not sufficiently pure to draw any definite conclusions about whether or not noradrenaline is stored together with adenosine 5'-triphosphate in small noradrenergic vesicles.